Version #6, 7/15/2004. Formatted by Tim Hawes, Northeastern University, Boston.
This version was prepared as a convenient reference document for people to discuss by referring to specific numbered words and word sequences. Done under the sponsorship of Bob Futrelle, Northeastern U. Each sentence is sequentially numbered. All words and punctuation items in each sentence are also numbered. The tokenization breaks apart many items that were contiguous in the original; this allows a more "fine-grained" discussion. The original article is available at this URL.
PLoS Biology Volume 2 | Issue 4 | APRIL 2004 Dissection and Design of Yeast Prions Lev Z. Osherovich, Brian S. Cox, Mick F. Tuite, and Jonathan S. Weissman
The aggregation of misfolded proteins underlies a diverse range of human diseases, including sporadic amyloidoses such as Alzheimer's disease and hereditary neuropathies such as Huntington's disease (Dobson 1999).
The2.1 aggregation2.2 of2.3 misfolded2.4 proteins2.5 underlies2.6 a2.7 diverse2.8 range2.9 of2.10 human2.11 diseases2.12,2.13 including2.14 sporadic2.15 amyloidoses2.16 such2.17 as2.18 Alzheimer's2.19 disease2.20 and2.21 hereditary2.22 neuropathies2.23 such2.24 as2.25 Huntington's2.26 disease2.27 (2.28Dobson2.29 19992.30)126.96.36.199
Prions are a special class of protein aggregates that replicate their conformation and spread infectiously (Prusiner 1998).
Prions3.1 are3.2 a3.3 special3.4 class3.5 of3.6 protein3.7 aggregates3.8 that3.9 replicate3.10 their3.11 conformation3.12 and3.13 spread3.14 infectiously3.15 (3.16Prusiner3.17 19983.18)188.8.131.52
After the discovery that prion aggregates are responsible for the mammalian transmissible spongiform encephalopathies, several epigenetically heritable traits in fungi were also found to depend on a prion mechanism (Wickner 1994; Uptain and Lindquist 2002; Osherovich and Weissman 2004).
After4.1 the4.2 discovery4.3 that4.4 prion4.5 aggregates4.6 are4.7 responsible4.8 for4.9 the4.10 mammalian4.11 transmissible4.12 spongiform4.13 encephalopathies4.14,4.15 several4.16 epigenetically4.17 heritable4.18 traits4.19 in4.20 fungi4.21 were4.22 also4.23 found4.24 to4.25 depend4.26 on4.27 a4.28 prion4.29 mechanism4.30 (4.31Wickner4.32 19944.33;4.34 Uptain4.35 and4.36 Lindquist4.37 20024.38;4.39 Osherovich4.40 and4.41 Weissman4.42 20044.43)184.108.40.206
In Saccharomyces cerevisiae and Podospora anserina, prions are transmitted from cell to cell through mating and cell division, resulting in readily assayed phenotypes with a non-Mendelian pattern of inheritance (Liebman and Derkatch 1999).
In5.1 Saccharomyces5.2 cerevisiae5.3 and5.4 Podospora5.5 anserina5.6,5.7 prions5.8 are5.9 transmitted5.10 from5.11 cell5.12 to5.13 cell5.14 through5.15 mating5.16 and5.17 cell5.18 division5.19,5.20 resulting5.21 in5.22 readily5.23 assayed5.24 phenotypes5.25 with5.26 a5.27 non-Mendelian5.28 pattern5.29 of5.30 inheritance5.31 (5.32Liebman5.33 and5.34 Derkatch5.35 19995.36)220.127.116.11
The yeast non-Mendelian factors [PSI+] (Cox 1965) and [URE3] (Lacroute 1971), which are prion forms of the translation termination factor Sup35p and the transcriptional activator Ure2p, respectively, have served as useful models for the formation and replication of heritable protein aggregates.
The6.1 yeast6.2 non-Mendelian6.3 factors6.4 [6.5PSI6.6+6.7]6.8 (6.9Cox6.10 19656.11)6.12 and6.13 [6.14URE36.15]6.16 (6.17Lacroute6.18 19716.19)6.20,6.21 which6.22 are6.23 prion6.24 forms6.25 of6.26 the6.27 translation6.28 termination6.29 factor6.30 Sup35p6.31 and6.32 the6.33 transcriptional6.34 activator6.35 Ure2p6.36,6.37 respectively6.38,6.39 have6.40 served6.41 as6.42 useful6.43 models6.44 for6.45 the6.46 formation6.47 and6.48 replication6.49 of6.50 heritable6.51 protein6.52 aggregates18.104.22.168
Prion forms of Sup35p and Ure2p lead to defects in their respective biochemical activities (translation termination and nitrogen catabolite repression).
Prion7.1 forms7.2 of7.3 Sup35p7.4 and7.5 Ure2p7.6 lead7.7 to7.8 defects7.9 in7.10 their7.11 respective7.12 biochemical7.13 activities7.14 (7.15translation7.16 termination7.17 and7.18 nitrogen7.19 catabolite7.20 repression7.21)22.214.171.124
Mutational analysis has shown the glutamine/asparagine-rich (Q/N-rich) amino-terminal (N) domains of these proteins to be critical for prion behavior (Ter-Avanesyan et al. 1993; Masison and Wickner 1995; Patino et al. 1996; Paushkin et al. 1996; DePace et al. 1998).
Mutational8.1 analysis8.2 has8.3 shown8.4 the8.5 glutamine/asparagine-rich8.6 (8.7Q/N-rich8.8)8.9 amino-terminal8.10 (8.11N8.12)8.13 domains8.14 of8.15 these8.16 proteins8.17 to8.18 be8.19 critical8.20 for8.21 prion8.22 behavior8.23 (8.24Ter-Avanesyan8.25 et8.26 al126.96.36.199 19938.29;8.30 Masison8.31 and8.32 Wickner8.33 19958.34;8.35 Patino8.36 et8.37 al188.8.131.52 19968.40;8.41 Paushkin8.42 et8.43 al184.108.40.206 19968.46;8.47 DePace8.48 et8.49 al220.127.116.11 19988.52)18.104.22.168
In vitro, these Q/N-rich domains form self-seeding, Β-sheet-rich amyloid fibrils similar to those associated with Alzheimer's and Huntington's diseases (Glover et al. 1997; King et al. 1997; Taylor et al. 1999).
In9.1 vitro9.2,9.3 these9.4 Q/N-rich9.5 domains9.6 form9.7 self-seeding9.8,9.9 Β-sheet-rich9.10 amyloid9.11 fibrils9.12 similar9.13 to9.14 those9.15 associated9.16 with9.17 Alzheimer's9.18 and9.19 Huntington's9.20 diseases9.21 (9.22Glover9.23 et9.24 al22.214.171.124 19979.27;9.28 King9.29 et9.30 al126.96.36.199 19979.33;9.34 Taylor9.35 et9.36 al188.8.131.52 19999.39)184.108.40.206
The autocatalytic aggregation of yeast prion proteins often shows a high specificity for like molecules; for example, Sup35p N domains from different yeast species form prion aggregates that preferentially interact with molecules of their own kind (Santoso et al. 2000; Chernoff et al. 2000; Kushnirov et al. 2000; Zadorskii et al. 2000; Nakayashiki et al. 2001).
The10.1 autocatalytic10.2 aggregation10.3 of10.4 yeast10.5 prion10.6 proteins10.7 often10.8 shows10.9 a10.10 high10.11 specificity10.12 for10.13 like10.14 molecules10.15;10.16 for10.17 example10.18,10.19 Sup35p10.20 N10.21 domains10.22 from10.23 different10.24 yeast10.25 species10.26 form10.27 prion10.28 aggregates10.29 that10.30 preferentially10.31 interact10.32 with10.33 molecules10.34 of10.35 their10.36 own10.37 kind10.38 (10.39Santoso10.40 et10.41 al10.42.10.43 200010.44;10.45 Chernoff10.46 et10.47 al10.48.10.49 200010.50;10.51 Kushnirov10.52 et10.53 al10.54.10.55 200010.56;10.57 Zadorskii10.58 et10.59 al10.60.10.61 200010.62;10.63 Nakayashiki10.64 et10.65 al10.66.10.67 200110.68)10.69.10.70
[PSI+] and [URE3] can be eliminated by transient growth in the presence of guanidine hydrochloride (GuHCl), which “cures” cells of prions by inhibiting Hsp104p, a molecular chaperone needed for prion replication (Chernoff et al. 1995; Jung et al. 2002; Ness et al. 2002).
[11.1PSI11.2+11.3]11.4 and11.5 [11.6URE311.7]11.8 can11.9 be11.10 eliminated11.11 by11.12 transient11.13 growth11.14 in11.15 the11.16 presence11.17 of11.18 guanidine11.19 hydrochloride11.20 (11.21GuHCl11.22)11.23,11.24 which11.25 “11.26cures11.27”11.28 cells11.29 of11.30 prions11.31 by11.32 inhibiting11.33 Hsp104p11.34,11.35 a11.36 molecular11.37 chaperone11.38 needed11.39 for11.40 prion11.41 replication11.42 (11.43Chernoff11.44 et11.45 al220.127.116.11 199511.48;11.49 Jung11.50 et11.51 al18.104.22.168 200211.54;11.55 Ness11.56 et11.57 al22.214.171.124 200211.60)126.96.36.199
A surprisingly large number of proteins in S. cerevisiae and other eukaryotes have lengthy Q/N-rich tracts ostensibly similar to those found in the prion-forming domains of Sup35p and Ure2p (Michelitsch and Weissman 2000).
A12.1 surprisingly12.2 large12.3 number12.4 of12.5 proteins12.6 in12.7 S188.8.131.52 cerevisiae12.10 and12.11 other12.12 eukaryotes12.13 have12.14 lengthy12.15 Q/N-rich12.16 tracts12.17 ostensibly12.18 similar12.19 to12.20 those12.21 found12.22 in12.23 the12.24 prion-forming12.25 domains12.26 of12.27 Sup35p12.28 and12.29 Ure2p12.30 (12.31Michelitsch12.32 and12.33 Weissman12.34 200012.35)184.108.40.206
From among these, we and another group identified two novel proteins, New1p and Rnq1p, with prion-forming domains resembling those of Sup35p and Ure2p (Santoso et al. 2000; Sondheimer and Lindquist 2000).
From13.1 among13.2 these13.3,13.4 we13.5 and13.6 another13.7 group13.8 identified13.9 two13.10 novel13.11 proteins13.12,13.13 New1p13.14 and13.15 Rnq1p13.16,13.17 with13.18 prion-forming13.19 domains13.20 resembling13.21 those13.22 of13.23 Sup35p13.24 and13.25 Ure2p13.26 (13.27Santoso13.28 et13.29 al220.127.116.11 200013.32;13.33 Sondheimer13.34 and13.35 Lindquist13.36 200013.37)18.104.22.168
When these Q/N-rich domains were fused to green fluorescent protein (GFP) and overexpressed, they formed visible aggregates resembling those of GFP-labeled Sup35p in [PSI+] cells.
When14.1 these14.2 Q/N-rich14.3 domains14.4 were14.5 fused14.6 to14.7 green14.8 fluorescent14.9 protein14.10 (14.11GFP14.12)14.13 and14.14 overexpressed14.15,14.16 they14.17 formed14.18 visible14.19 aggregates14.20 resembling14.21 those14.22 of14.23 GFP-labeled14.24 Sup35p14.25 in14.26 [14.27PSI14.28+14.29]14.30 cells22.214.171.124
Fusion proteins in which these domains were introduced in place of the Sup35p prion domain could support distinct, self-specific prion states that recapitulated the translation termination defect associated with [PSI+].
Fusion15.1 proteins15.2 in15.3 which15.4 these15.5 domains15.6 were15.7 introduced15.8 in15.9 place15.10 of15.11 the15.12 Sup35p15.13 prion15.14 domain15.15 could15.16 support15.17 distinct15.18,15.19 self-specific15.20 prion15.21 states15.22 that15.23 recapitulated15.24 the15.25 translation15.26 termination15.27 defect15.28 associated15.29 with15.30 [15.31PSI15.32+15.33]126.96.36.199
Rnq1p was later shown to underlie a naturally occurring prion called [PIN+], which promotes the aggregation of Q/N-rich proteins such as Sup35p; overexpressed Sup35p forms aggregates and stimulates the appearance of [PSI+] only in [PIN+] strains (Derkatch et al. 1997; Derkatch et al. 2001).
Rnq1p16.1 was16.2 later16.3 shown16.4 to16.5 underlie16.6 a16.7 naturally16.8 occurring16.9 prion16.10 called16.11 [16.12PIN16.13+16.14]16.15,16.16 which16.17 promotes16.18 the16.19 aggregation16.20 of16.21 Q/N-rich16.22 proteins16.23 such16.24 as16.25 Sup35p16.26;16.27 overexpressed16.28 Sup35p16.29 forms16.30 aggregates16.31 and16.32 stimulates16.33 the16.34 appearance16.35 of16.36 [16.37PSI16.38+16.39]16.40 only16.41 in16.42 [16.43PIN16.44+16.45]16.46 strains16.47 (16.48Derkatch16.49 et16.50 al188.8.131.52 199716.53;16.54 Derkatch16.55 et16.56 al184.108.40.206 200116.59)220.127.116.11
Aggregates of the New1p prion domain, whether resulting from overexpression or from a constitutive prion form (termed [NU+]), also promoted the aggregation of other Q/N-rich proteins, emulating the effect of [PIN+] (Osherovich and Weissman 2001).
Aggregates17.1 of17.2 the17.3 New1p17.4 prion17.5 domain17.6,17.7 whether17.8 resulting17.9 from17.10 overexpression17.11 or17.12 from17.13 a17.14 constitutive17.15 prion17.16 form17.17 (17.18termed17.19 [17.20NU17.21+17.22]17.23)17.24,17.25 also17.26 promoted17.27 the17.28 aggregation17.29 of17.30 other17.31 Q/N-rich17.32 proteins17.33,17.34 emulating17.35 the17.36 effect17.37 of17.38 [17.39PIN17.40+17.41]17.42 (17.43Osherovich17.44 and17.45 Weissman17.46 200117.47)18.104.22.168
Schematic Diagram of Sup35p and New1pPrion domains of both proteins are enlarged in the center, highlighting the Q/N-rich tract of Sup35p (blue), the NYN tripeptide repeat of New1p (purple), and the oligopeptide repeat sequences (orange) found in both proteins.
Schematic19.1 Diagram19.2 of19.3 Sup35p19.4 and19.5 New1pPrion19.6 domains19.7 of19.8 both19.9 proteins19.10 are19.11 enlarged19.12 in19.13 the19.14 center19.15,19.16 highlighting19.17 the19.18 Q/N-rich19.19 tract19.20 of19.21 Sup35p19.22 (19.23blue19.24)19.25,19.26 the19.27 NYN19.28 tripeptide19.29 repeat19.30 of19.31 New1p19.32 (19.33purple19.34)19.35,19.36 and19.37 the19.38 oligopeptide19.39 repeat19.40 sequences19.41 (19.42orange19.43)19.44 found19.45 in19.46 both19.47 proteins22.214.171.124
The sequence of the NEW1 oligopetide repeat (residues 50–70) is QQQRNWKQGGNYQQGGYQSYN, while that of the adjacent tripeptide repeat region (residues 71–100) is SNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ.
The20.1 sequence20.2 of20.3 the20.4 NEW120.5 oligopetide20.6 repeat20.7 (20.8residues20.9 5020.10–20.117020.12)20.13 is20.14 QQQRNWKQGGNYQQGGYQSYN20.15,20.16 while20.17 that20.18 of20.19 the20.20 adjacent20.21 tripeptide20.22 repeat20.23 region20.24 (20.25residues20.26 7120.27–20.2810020.29)20.30 is20.31 SNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ126.96.36.199
Many sequences with Q/N content as high as that of Sup35p and Ure2p, including human polyglutamine expansion disease proteins, form visible aggregates when overexpressed in yeast as GFP fusions (Krobitsch and Lindquist 2000; Osherovich and Weissman 2001; Meriin et al. 2002).
Many21.1 sequences21.2 with21.3 Q/N21.4 content21.5 as21.6 high21.7 as21.8 that21.9 of21.10 Sup35p21.11 and21.12 Ure2p21.13,21.14 including21.15 human21.16 polyglutamine21.17 expansion21.18 disease21.19 proteins21.20,21.21 form21.22 visible21.23 aggregates21.24 when21.25 overexpressed21.26 in21.27 yeast21.28 as21.29 GFP21.30 fusions21.31 (21.32Krobitsch21.33 and21.34 Lindquist21.35 200021.36;21.37 Osherovich21.38 and21.39 Weissman21.40 200121.41;21.42 Meriin21.43 et21.44 al188.8.131.52 200221.47)184.108.40.206
However, only a limited number of Q/N-rich sequences are bone fide prion domains capable of propagating these aggregates over multiple cell generations even when expressed at low levels (J. Hood and J.S.W, unpublished data).
However22.1,22.2 only22.3 a22.4 limited22.5 number22.6 of22.7 Q/N-rich22.8 sequences22.9 are22.10 bone22.11 fide22.12 prion22.13 domains22.14 capable22.15 of22.16 propagating22.17 these22.18 aggregates22.19 over22.20 multiple22.21 cell22.22 generations22.23 even22.24 when22.25 expressed22.26 at22.27 low22.28 levels22.29 (22.30J220.127.116.11 Hood22.33 and22.34 J18.104.22.168S22.214.171.124W22.39,22.40 unpublished22.41 data22.42)126.96.36.199
To understand what distinguishes generic Q/N-rich aggregates from heritable prions, we conducted a detailed dissection of the prion-forming regions of Sup35p and New1p.
To23.1 understand23.2 what23.3 distinguishes23.4 generic23.5 Q/N-rich23.6 aggregates23.7 from23.8 heritable23.9 prions23.10,23.11 we23.12 conducted23.13 a23.14 detailed23.15 dissection23.16 of23.17 the23.18 prion-forming23.19 regions23.20 of23.21 Sup35p23.22 and23.23 New1p188.8.131.52
We found that the prion properties of Sup35p and New1p require the presence of two independent and portable sequence elements within their prion domains.
We24.1 found24.2 that24.3 the24.4 prion24.5 properties24.6 of24.7 Sup35p24.8 and24.9 New1p24.10 require24.11 the24.12 presence24.13 of24.14 two24.15 independent24.16 and24.17 portable24.18 sequence24.19 elements24.20 within24.21 their24.22 prion24.23 domains184.108.40.206
One element mediates the growth of prion aggregates by incorporation of soluble monomers.
One25.1 element25.2 mediates25.3 the25.4 growth25.5 of25.6 prion25.7 aggregates25.8 by25.9 incorporation25.10 of25.11 soluble25.12 monomers220.127.116.11
The second promotes the inheritance of aggregates, generating new heritable “seeds” which can be partitioned between mother and daughter cells during cell division.
The26.1 second26.2 promotes26.3 the26.4 inheritance26.5 of26.6 aggregates26.7,26.8 generating26.9 new26.10 heritable26.11 “26.12seeds26.13”26.14 which26.15 can26.16 be26.17 partitioned26.18 between26.19 mother26.20 and26.21 daughter26.22 cells26.23 during26.24 cell26.25 division18.104.22.168
Distinct Regions of the New1p Prion Domain Mediate Prion Growth and Division
Distinct28.1 Regions28.2 of28.3 the28.4 New1p28.5 Prion28.6 Domain28.7 Mediate28.8 Prion28.9 Growth28.10 and28.11 Division28.12
Sup35p can alternate between a biochemically active, soluble form ([psi–]) and an aggregated prion state ([PSI+]) with diminished translation termination activity, which can be monitored by nonsense suppression of the mutant ade1–14 allele (Liebman and Derkatch 1999).
Sup35p29.1 can29.2 alternate29.3 between29.4 a29.5 biochemically29.6 active29.7,29.8 soluble29.9 form29.10 (29.11[29.12psi29.13–29.14]29.15)29.16 and29.17 an29.18 aggregated29.19 prion29.20 state29.21 (29.22[29.23PSI29.24+29.25]29.26)29.27 with29.28 diminished29.29 translation29.30 termination29.31 activity29.32,29.33 which29.34 can29.35 be29.36 monitored29.37 by29.38 nonsense29.39 suppression29.40 of29.41 the29.42 mutant29.43 ade129.44–29.451429.46 allele29.47 (29.48Liebman29.49 and29.50 Derkatch29.51 199929.52)22.214.171.124
Whereas [psi–] strains form red colonies on yeast extract-peptone-dextrose (YEPD) medium and cannot grow in the absence of adenine, [PSI+] strains suppress the premature stop codon in ade1-14, and thus appear pink or white on YEPD medium and grow on adenine-free medium (a phenotype termed adenine prototrophy, Ade+).
Whereas30.1 [30.2psi30.3–30.4]30.5 strains30.6 form30.7 red30.8 colonies30.9 on30.10 yeast30.11 extract-peptone-dextrose30.12 (30.13YEPD30.14)30.15 medium30.16 and30.17 cannot30.18 grow30.19 in30.20 the30.21 absence30.22 of30.23 adenine30.24,30.25 [30.26PSI30.27+30.28]30.29 strains30.30 suppress30.31 the30.32 premature30.33 stop30.34 codon30.35 in30.36 ade1-1430.37,30.38 and30.39 thus30.40 appear30.41 pink30.42 or30.43 white30.44 on30.45 YEPD30.46 medium30.47 and30.48 grow30.49 on30.50 adenine-free30.51 medium30.52 (30.53a30.54 phenotype30.55 termed30.56 adenine30.57 prototrophy30.58,30.59 Ade30.60+30.61)126.96.36.199
The N or prion domain of Sup35p (residues 1-112) is required for [PSI+] formation but is dispensable for the translation termination activity of the carboxy-terminal C domain (Ter-Avanesyan et al. 1993).
The31.1 N31.2 or31.3 prion31.4 domain31.5 of31.6 Sup35p31.7 (31.8residues31.9 1-11231.10)31.11 is31.12 required31.13 for31.14 [31.15PSI31.16+31.17]31.18 formation31.19 but31.20 is31.21 dispensable31.22 for31.23 the31.24 translation31.25 termination31.26 activity31.27 of31.28 the31.29 carboxy-terminal31.30 C31.31 domain31.32 (31.33Ter-Avanesyan31.34 et31.35 al188.8.131.52 199331.38)184.108.40.206
The charged middle domain (M) is not required for prion behavior, but modulates the efficiency of chaperone-dependent prion transmission (Liu et al. 2002; L.Z.O., unpublished data) (Figure 1).
The32.1 charged32.2 middle32.3 domain32.4 (32.5M32.6)32.7 is32.8 not32.9 required32.10 for32.11 prion32.12 behavior32.13,32.14 but32.15 modulates32.16 the32.17 efficiency32.18 of32.19 chaperone-dependent32.20 prion32.21 transmission32.22 (32.23Liu32.24 et32.25 al220.127.116.11 200232.28;32.29 L18.104.22.168Z22.214.171.124O126.96.36.199,32.36 unpublished32.37 data32.38)32.39 (32.40Figure32.41 132.42)188.8.131.52
Two distinct regions in the N domain have previously been implicated in Sup35p aggregation: a Q/N-rich tract (residues 1–39) (DePace et al. 1998) and an oligopeptide repeat (residues 40–112) that consists of five and a half degenerate repeats of the consensus sequence P/QQGGYQQ/SYN (Liu and Lindquist 1999; Parham et al. 2001; Crist et al. 2003).
Two33.1 distinct33.2 regions33.3 in33.4 the33.5 N33.6 domain33.7 have33.8 previously33.9 been33.10 implicated33.11 in33.12 Sup35p33.13 aggregation33.14:33.15 a33.16 Q/N-rich33.17 tract33.18 (33.19residues33.20 133.21–33.223933.23)33.24 (33.25DePace33.26 et33.27 al184.108.40.206 199833.30)33.31 and33.32 an33.33 oligopeptide33.34 repeat33.35 (33.36residues33.37 4033.38–33.3911233.40)33.41 that33.42 consists33.43 of33.44 five33.45 and33.46 a33.47 half33.48 degenerate33.49 repeats33.50 of33.51 the33.52 consensus33.53 sequence33.54 P/QQGGYQQ/SYN33.55 (33.56Liu33.57 and33.58 Lindquist33.59 199933.60;33.61 Parham33.62 et33.63 al220.127.116.11 200133.66;33.67 Crist33.68 et33.69 al18.104.22.168 200333.72)22.214.171.124
We had earlier identified New1p as an uncharacterized protein with a Sup35p-like N-terminal domain; when fused to the M and C domains of Sup35p, the first 153 residues of New1p (New11–153) supported a [PSI+]-like prion state termed [NU+] (Santoso et al. 2000).
We34.1 had34.2 earlier34.3 identified34.4 New1p34.5 as34.6 an34.7 uncharacterized34.8 protein34.9 with34.10 a34.11 Sup35p-like34.12 N-terminal34.13 domain34.14;34.15 when34.16 fused34.17 to34.18 the34.19 M34.20 and34.21 C34.22 domains34.23 of34.24 Sup35p34.25,34.26 the34.27 first34.28 15334.29 residues34.30 of34.31 New1p34.32 (34.33New1134.34–34.3515334.36)34.37 supported34.38 a34.39 [34.40PSI34.41+34.42]34.43-like34.44 prion34.45 state34.46 termed34.47 [34.48NU34.49+34.50]34.51 (34.52Santoso34.53 et34.54 al126.96.36.199 200034.57)188.8.131.52
Sup35p and New1p have regions of clear similarity beyond their high Q/N content (Figure 1).
Sup35p35.1 and35.2 New1p35.3 have35.4 regions35.5 of35.6 clear35.7 similarity35.8 beyond35.9 their35.10 high35.11 Q/N35.12 content35.13 (35.14Figure35.15 135.16)184.108.40.206
The prion domains of both have Q/N-rich tracts and oligopeptide repeat regions, although their order is reversed.
The36.1 prion36.2 domains36.3 of36.4 both36.5 have36.6 Q/N-rich36.7 tracts36.8 and36.9 oligopeptide36.10 repeat36.11 regions36.12,36.13 although36.14 their36.15 order36.16 is36.17 reversed220.127.116.11
The C-terminal domains of New1p and Sup35p also appear to be related, based on modest homology and the similarity of the translation termination defects in sup35 (Song and Liebman 1985) and new1 mutants (L.Z.O., unpublished data).
The37.1 C-terminal37.2 domains37.3 of37.4 New1p37.5 and37.6 Sup35p37.7 also37.8 appear37.9 to37.10 be37.11 related37.12,37.13 based37.14 on37.15 modest37.16 homology37.17 and37.18 the37.19 similarity37.20 of37.21 the37.22 translation37.23 termination37.24 defects37.25 in37.26 sup3537.27 (37.28Song37.29 and37.30 Liebman37.31 198537.32)37.33 and37.34 new137.35 mutants37.36 (37.37L18.104.22.168Z22.214.171.124O126.96.36.199,37.44 unpublished37.45 data37.46)188.8.131.52
To understand the sequence requirements for the prion behavior of New1p, we constructed a series of truncated prion domains (Figure 2A) and examined their participation in several critical steps of the prion replication cycle.
To38.1 understand38.2 the38.3 sequence38.4 requirements38.5 for38.6 the38.7 prion38.8 behavior38.9 of38.10 New1p38.11,38.12 we38.13 constructed38.14 a38.15 series38.16 of38.17 truncated38.18 prion38.19 domains38.20 (38.21Figure38.22 2A38.23)38.24 and38.25 examined38.26 their38.27 participation38.28 in38.29 several38.30 critical38.31 steps38.32 of38.33 the38.34 prion38.35 replication38.36 cycle184.108.40.206
We first asked whether these truncated prion domains could form visible foci when fused to GFP (aggregation).
We39.1 first39.2 asked39.3 whether39.4 these39.5 truncated39.6 prion39.7 domains39.8 could39.9 form39.10 visible39.11 foci39.12 when39.13 fused39.14 to39.15 GFP39.16 (39.17aggregation39.18)220.127.116.11
Next, we examined whether such aggregates could convert New11–153 into a [NU+] prion state (induction).
Next40.1,40.2 we40.3 examined40.4 whether40.5 such40.6 aggregates40.7 could40.8 convert40.9 New1140.10–40.1115340.12 into40.13 a40.14 [40.15NU40.16+40.17]40.18 prion40.19 state40.20 (40.21induction40.22)18.104.22.168
Finally, we fused these constructs to the M and C domains of Sup35p (–M-C), introduced them in place of endogenous SUP35, and assessed whether these proteins could adopt stable prion states (maintenance).
Finally41.1,41.2 we41.3 fused41.4 these41.5 constructs41.6 to41.7 the41.8 M41.9 and41.10 C41.11 domains41.12 of41.13 Sup35p41.14 (41.15–41.16M-C41.17)41.18,41.19 introduced41.20 them41.21 in41.22 place41.23 of41.24 endogenous41.25 SUP3541.26,41.27 and41.28 assessed41.29 whether41.30 these41.31 proteins41.32 could41.33 adopt41.34 stable41.35 prion41.36 states41.37 (41.38maintenance41.39)22.214.171.124
We found that distinct regions within the New1p prion domain are necessary for the induction and maintenance of [NU+] (Figure 2A).
We42.1 found42.2 that42.3 distinct42.4 regions42.5 within42.6 the42.7 New1p42.8 prion42.9 domain42.10 are42.11 necessary42.12 for42.13 the42.14 induction42.15 and42.16 maintenance42.17 of42.18 [42.19NU42.20+42.21]42.22 (42.23Figure42.24 2A42.25)126.96.36.199
The asparagine-tyrosine-asparagine (NYN) repeat (residues 70–100), which we had earlier shown to be sufficient for aggregation (Osherovich and Weissman 2001), also proved sufficient for induction of [NU+].
The43.1 asparagine-tyrosine-asparagine43.2 (43.3NYN43.4)43.5 repeat43.6 (43.7residues43.8 7043.9–43.1010043.11)43.12,43.13 which43.14 we43.15 had43.16 earlier43.17 shown43.18 to43.19 be43.20 sufficient43.21 for43.22 aggregation43.23 (43.24Osherovich43.25 and43.26 Weissman43.27 200143.28)43.29,43.30 also43.31 proved43.32 sufficient43.33 for43.34 induction43.35 of43.36 [43.37NU43.38+43.39]188.8.131.52
As with the full-length New1p prion domain, overexpression of the NYN repeat efficiently stimulated the appearance of Ade+ in [nu–] cells (Figure 2B, left).
As44.1 with44.2 the44.3 full-length44.4 New1p44.5 prion44.6 domain44.7,44.8 overexpression44.9 of44.10 the44.11 NYN44.12 repeat44.13 efficiently44.14 stimulated44.15 the44.16 appearance44.17 of44.18 Ade44.19+44.20 in44.21 [44.22nu44.23–44.24]44.25 cells44.26 (44.27Figure44.28 2B44.29,44.30 left44.31)184.108.40.206
However, stable prion maintenance required both the NYN repeat and the adjacent oligopeptide repeat.
However45.1,45.2 stable45.3 prion45.4 maintenance45.5 required45.6 both45.7 the45.8 NYN45.9 repeat45.10 and45.11 the45.12 adjacent45.13 oligopeptide45.14 repeat220.127.116.11
In a strain with this minimized New1p prion domain (residues 50–100), overexpression of the full prion domain or of the NYN repeat alone promoted the appearance of Ade+ colonies (Figure 2B, right).
In46.1 a46.2 strain46.3 with46.4 this46.5 minimized46.6 New1p46.7 prion46.8 domain46.9 (46.10residues46.11 5046.12–46.1310046.14)46.15,46.16 overexpression46.17 of46.18 the46.19 full46.20 prion46.21 domain46.22 or46.23 of46.24 the46.25 NYN46.26 repeat46.27 alone46.28 promoted46.29 the46.30 appearance46.31 of46.32 Ade46.33+46.34 colonies46.35 (46.36Figure46.37 2B46.38,46.39 right46.40)18.104.22.168
The resulting convertants remained Ade+ after loss of the inducer plasmid but reverted to Ade- after transient GuHCl treatment (Figure 2C).
The47.1 resulting47.2 convertants47.3 remained47.4 Ade47.5+47.6 after47.7 loss47.8 of47.9 the47.10 inducer47.11 plasmid47.12 but47.13 reverted47.14 to47.15 Ade- after47.16 transient47.17 GuHCl47.18 treatment47.19 (47.20Figure47.21 2C47.22)22.214.171.124
We conclude that the oligopeptide repeat and the NYN repeat of New1p together are sufficient to support a prion state, termed [NU+]mini, which recapitulates the characteristics of [NU+].
We48.1 conclude48.2 that48.3 the48.4 oligopeptide48.5 repeat48.6 and48.7 the48.8 NYN48.9 repeat48.10 of48.11 New1p48.12 together48.13 are48.14 sufficient48.15 to48.16 support48.17 a48.18 prion48.19 state48.20,48.21 termed48.22 [48.23NU48.24+48.25]48.26mini48.27,48.28 which48.29 recapitulates48.30 the48.31 characteristics48.32 of48.33 [48.34NU48.35+48.36]126.96.36.199
Dissection of the New1p Prion Domain Reveals Distinct Regions Responsible for Aggregation and Prion Inheritance
Dissection50.1 of50.2 the50.3 New1p50.4 Prion50.5 Domain50.6 Reveals50.7 Distinct50.8 Regions50.9 Responsible50.10 for50.11 Aggregation50.12 and50.13 Prion50.14 Inheritance50.15
(A) Indicated fragments of New1p (left) were expressed as GFP fusions (inducers) in a [nu–] [pin–] strain, examined by microscopy for GFP aggregation, then plated on SD-ade medium to assess induction of [NU+].
(51.1A51.2)51.3 Indicated51.4 fragments51.5 of51.6 New1p51.7 (51.8left51.9)51.10 were51.11 expressed51.12 as51.13 GFP51.14 fusions51.15 (51.16inducers51.17)51.18 in51.19 a51.20 [51.21nu51.22–51.23]51.24 [51.25pin51.26–51.27]51.28 strain51.29,51.30 examined51.31 by51.32 microscopy51.33 for51.34 GFP51.35 aggregation51.36,51.37 then51.38 plated51.39 on51.40 SD-ade51.41 medium51.42 to51.43 assess51.44 induction51.45 of51.46 [51.47NU51.48+51.49]188.8.131.52
The symbol “+” indicates induction frequencies of at least 5%; the symbol “–” indicates no induction.
The52.1 symbol52.2 “52.3+52.4”52.5 indicates52.6 induction52.7 frequencies52.8 of52.9 at52.10 least52.11 5%;52.12 the52.13 symbol52.14 “52.15–52.16”52.17 indicates52.18 no52.19 induction184.108.40.206
Maintenance was assessed by the ability of an episomal maintainer version of the indicated fragment to support an Ade+ state after overexpression of New11–153-GFP (see Materials and Methods).
Maintenance53.1 was53.2 assessed53.3 by53.4 the53.5 ability53.6 of53.7 an53.8 episomal53.9 maintainer53.10 version53.11 of53.12 the53.13 indicated53.14 fragment53.15 to53.16 support53.17 an53.18 Ade53.19+53.20 state53.21 after53.22 overexpression53.23 of53.24 New1153.25–53.26153-GFP53.27 (53.28see53.29 Materials53.30 and53.31 Methods53.32)220.127.116.11
The aggregation of New1-GFP fusions (second column) has been previously reported (Osherovich and Weissman 2001).
The54.1 aggregation54.2 of54.3 New1-GFP54.4 fusions54.5 (54.6second54.7 column54.8)54.9 has54.10 been54.11 previously54.12 reported54.13 (54.14Osherovich54.15 and54.16 Weissman54.17 200154.18)18.104.22.168
(B) The NYN repeat of New1p induces [NU+] and [NU+]mini.
(55.1B55.2)55.3 The55.4 NYN55.5 repeat55.6 of55.7 New1p55.8 induces55.9 [55.10NU55.11+55.12]55.13 and55.14 [55.15NU55.16+55.17]55.18mini22.214.171.124
New170–100-GFP was overexpressed in [nu–] and [nu–]mini strains ([pin–] and [PIN+] derivatives of each), along with vector only or New11–153-GFP controls.
New17056.1–56.2100-GFP56.3 was56.4 overexpressed56.5 in56.6 [56.7nu56.8–56.9]56.10 and56.11 [56.12nu56.13–56.14]56.15mini56.16 strains56.17 (56.18[56.19pin56.20–56.21]56.22 and56.23 [56.24PIN56.25+56.26]56.27 derivatives56.28 of56.29 each56.30)56.31,56.32 along56.33 with56.34 vector56.35 only56.36 or56.37 New1156.38–56.39153-GFP56.40 controls126.96.36.199
Averages of three independent trials, representing 600–2000 colonies, are shown for most induction experiments; inductions using New170–100-GFP were conducted twice.
Averages57.1 of57.2 three57.3 independent57.4 trials57.5,57.6 representing57.7 60057.8–57.9200057.10 colonies57.11,57.12 are57.13 shown57.14 for57.15 most57.16 induction57.17 experiments57.18;57.19 inductions57.20 using57.21 New17057.22–57.23100-GFP57.24 were57.25 conducted57.26 twice188.8.131.52
Error bars show minimal and maximal observed induction efficiencies.
Error58.1 bars58.2 show58.3 minimal58.4 and58.5 maximal58.6 observed58.7 induction58.8 efficiencies184.108.40.206
(C) Reversibility of [NU+]mini.
(59.1C59.2)59.3 Reversibility59.4 of59.5 [59.6NU59.7+59.8]59.9mini220.127.116.11
The [pin–] Ade+ convertants obtained in (B) were colony purified on SD-ade medium and confirmed to have lost the inducer plasmid.
The60.1 [60.2pin60.3–60.4]60.5 Ade60.6+60.7 convertants60.8 obtained60.9 in60.10 (60.11B60.12)60.13 were60.14 colony60.15 purified60.16 on60.17 SD-ade60.18 medium60.19 and60.20 confirmed60.21 to60.22 have60.23 lost60.24 the60.25 inducer60.26 plasmid18.104.22.168
A stable [NU+]mini isolate is shown before and after induction, as well as after GuHCl treatment, along with [nu–] and [NU+] reference strains.
A61.1 stable61.2 [61.3NU61.4+61.5]61.6mini61.7 isolate61.8 is61.9 shown61.10 before61.11 and61.12 after61.13 induction61.14,61.15 as61.16 well61.17 as61.18 after61.19 GuHCl61.20 treatment61.21,61.22 along61.23 with61.24 [61.25nu61.26–61.27]61.28 and61.29 [61.30NU61.31+61.32]61.33 reference61.34 strains22.214.171.124
Dissection of the Sup35p Prion Domain
Dissection62.1 of62.2 the62.3 Sup35p62.4 Prion62.5 Domain62.6
In light of the similarity between New1p and Sup35p prion domains, we asked whether separate regions of Sup35p were involved in the induction and maintenance of [PSI+] aggregates (Figure 3).
In63.1 light63.2 of63.3 the63.4 similarity63.5 between63.6 New1p63.7 and63.8 Sup35p63.9 prion63.10 domains63.11,63.12 we63.13 asked63.14 whether63.15 separate63.16 regions63.17 of63.18 Sup35p63.19 were63.20 involved63.21 in63.22 the63.23 induction63.24 and63.25 maintenance63.26 of63.27 [63.28PSI63.29+63.30]63.31 aggregates63.32 (63.33Figure63.34 363.35)126.96.36.199
We constructed a series of truncated Sup35p N domains and analyzed their behavior in the aggregation, induction, and maintenance assays described above for [NU+].
We64.1 constructed64.2 a64.3 series64.4 of64.5 truncated64.6 Sup35p64.7 N64.8 domains64.9 and64.10 analyzed64.11 their64.12 behavior64.13 in64.14 the64.15 aggregation64.16,64.17 induction64.18,64.19 and64.20 maintenance64.21 assays64.22 described64.23 above64.24 for64.25 [64.26NU64.27+64.28]188.8.131.52
Additionally, we examined the ability of truncated N domains to decorate preexisting Sup35p aggregates in [PSI+] strains.
Additionally65.1,65.2 we65.3 examined65.4 the65.5 ability65.6 of65.7 truncated65.8 N65.9 domains65.10 to65.11 decorate65.12 preexisting65.13 Sup35p65.14 aggregates65.15 in65.16 [65.17PSI65.18+65.19]65.20 strains184.108.40.206
We found that the Q/N-rich tract and a small portion of the adjacent oligopeptide repeat are responsible for Sup35p aggregation and de novo [PSI+] induction.
We66.1 found66.2 that66.3 the66.4 Q/N-rich66.5 tract66.6 and66.7 a66.8 small66.9 portion66.10 of66.11 the66.12 adjacent66.13 oligopeptide66.14 repeat66.15 are66.16 responsible66.17 for66.18 Sup35p66.19 aggregation66.20 and66.21 de66.22 novo66.23 [66.24PSI66.25+66.26]66.27 induction220.127.116.11
Deletions within the Q/N-rich tract or of oligopeptide repeat 1 abolished these properties, whereas a construct containing only the Q/N-rich region and the first two oligopeptide repeats (residues 1–64) aggregated and induced [PSI+] at levels comparable to the full prion domain, in agreement with King (2001).
Deletions67.1 within67.2 the67.3 Q/N-rich67.4 tract67.5 or67.6 of67.7 oligopeptide67.8 repeat67.9 167.10 abolished67.11 these67.12 properties67.13,67.14 whereas67.15 a67.16 construct67.17 containing67.18 only67.19 the67.20 Q/N-rich67.21 region67.22 and67.23 the67.24 first67.25 two67.26 oligopeptide67.27 repeats67.28 (67.29residues67.30 167.31–67.326467.33)67.34 aggregated67.35 and67.36 induced67.37 [67.38PSI67.39+67.40]67.41 at67.42 levels67.43 comparable67.44 to67.45 the67.46 full67.47 prion67.48 domain67.49,67.50 in67.51 agreement67.52 with67.53 King67.54 (67.55200167.56)18.104.22.168
A construct (residues 1–57) with a partial deletion of oligopeptide repeat 2 could still aggregate and induce [PSI+], albeit with decreased efficiency.
A68.1 construct68.2 (68.3residues68.4 168.5–68.65768.7)68.8 with68.9 a68.10 partial68.11 deletion68.12 of68.13 oligopeptide68.14 repeat68.15 268.16 could68.17 still68.18 aggregate68.19 and68.20 induce68.21 [68.22PSI68.23+68.24]68.25,68.26 albeit68.27 with68.28 decreased68.29 efficiency22.214.171.124
Although a construct lacking oligopeptide repeat 2 entirely (residues 1–49) did not induce [PSI+] de novo, this GFP fusion could nonetheless decorate preexisting Sup35p aggregates.
Although69.1 a69.2 construct69.3 lacking69.4 oligopeptide69.5 repeat69.6 269.7 entirely69.8 (69.9residues69.10 169.11–69.124969.13)69.14 did69.15 not69.16 induce69.17 [69.18PSI69.19+69.20]69.21 de69.22 novo69.23,69.24 this69.25 GFP69.26 fusion69.27 could69.28 nonetheless69.29 decorate69.30 preexisting69.31 Sup35p69.32 aggregates126.96.36.199
Thus, while oligopeptide repeat 2 contributes to the aggregation of Sup35p, the primary determinants of prion induction reside in the amino-terminal Q/N-rich region and oligopeptide repeat 1.
Thus70.1,70.2 while70.3 oligopeptide70.4 repeat70.5 270.6 contributes70.7 to70.8 the70.9 aggregation70.10 of70.11 Sup35p70.12,70.13 the70.14 primary70.15 determinants70.16 of70.17 prion70.18 induction70.19 reside70.20 in70.21 the70.22 amino-terminal70.23 Q/N-rich70.24 region70.25 and70.26 oligopeptide70.27 repeat70.28 188.8.131.52
In contrast, the rest of the oligopeptide repeat region is needed for stable inheritance of [PSI+] aggregates.
In71.1 contrast71.2,71.3 the71.4 rest71.5 of71.6 the71.7 oligopeptide71.8 repeat71.9 region71.10 is71.11 needed71.12 for71.13 stable71.14 inheritance71.15 of71.16 [71.17PSI71.18+71.19]71.20 aggregates184.108.40.206
Constructs that did not form fluorescent foci could not retain [PSI+], suggesting that aggregation is a prerequisite for prion maintenance.
Constructs72.1 that72.2 did72.3 not72.4 form72.5 fluorescent72.6 foci72.7 could72.8 not72.9 retain72.10 [72.11PSI72.12+72.13]72.14,72.15 suggesting72.16 that72.17 aggregation72.18 is72.19 a72.20 prerequisite72.21 for72.22 prion72.23 maintenance220.127.116.11
However, aggregation is not sufficient for prion inheritance, as Sup35p constructs with deletions spanning oligopeptide repeats 3–5 could not support a prion state despite their ability to form aggregates and efficiently induce [PSI+].
However73.1,73.2 aggregation73.3 is73.4 not73.5 sufficient73.6 for73.7 prion73.8 inheritance73.9,73.10 as73.11 Sup35p73.12 constructs73.13 with73.14 deletions73.15 spanning73.16 oligopeptide73.17 repeats73.18 373.19–73.20573.21 could73.22 not73.23 support73.24 a73.25 prion73.26 state73.27 despite73.28 their73.29 ability73.30 to73.31 form73.32 aggregates73.33 and73.34 efficiently73.35 induce73.36 [73.37PSI73.38+73.39]18.104.22.168
Only the sixth (incomplete) oligopeptide repeat proved dispensable for [PSI+] maintenance, consistent with an earlier report (Parham et al. 2001).
Only74.1 the74.2 sixth74.3 (74.4incomplete74.5)74.6 oligopeptide74.7 repeat74.8 proved74.9 dispensable74.10 for74.11 [74.12PSI74.13+74.14]74.15 maintenance74.16,74.17 consistent74.18 with74.19 an74.20 earlier74.21 report74.22 (74.23Parham74.24 et74.25 al22.214.171.124 200174.28)126.96.36.199
The PNM2-1 Mutation in Oligopeptide Repeat 2 Specifically Compromises the Inheritance of [PSI+]
The75.1 PNM2-175.2 Mutation75.3 in75.4 Oligopeptide75.5 Repeat75.6 275.7 Specifically75.8 Compromises75.9 the75.10 Inheritance75.11 of75.12 [75.13PSI75.14+75.15]75.16
Our deletion analysis suggested that oligopeptide repeat 2 participated in both the formation and inheritance of Sup35p aggregates.
Our76.1 deletion76.2 analysis76.3 suggested76.4 that76.5 oligopeptide76.6 repeat76.7 276.8 participated76.9 in76.10 both76.11 the76.12 formation76.13 and76.14 inheritance76.15 of76.16 Sup35p76.17 aggregates188.8.131.52
We made use of a point mutation within oligopeptide repeat 2 known as PNM2-1 (G58D) to distinguish between these two functions.
We77.1 made77.2 use77.3 of77.4 a77.5 point77.6 mutation77.7 within77.8 oligopeptide77.9 repeat77.10 277.11 known77.12 as77.13 PNM2-177.14 (77.15G58D77.16)77.17 to77.18 distinguish77.19 between77.20 these77.21 two77.22 functions184.108.40.206
PNM2-1 (PSI No More) shows strong interference with [PSI+] in certain strain backgrounds through a poorly understood mechanism (McCready et al. 1977; Doel et al. 1994; Kochneva-Pervukhova et al. 1998; Derkatch et al. 1999).
PNM2-178.1 (78.2PSI78.3 No78.4 More78.5)78.6 shows78.7 strong78.8 interference78.9 with78.10 [78.11PSI78.12+78.13]78.14 in78.15 certain78.16 strain78.17 backgrounds78.18 through78.19 a78.20 poorly78.21 understood78.22 mechanism78.23 (78.24McCready78.25 et78.26 al220.127.116.11 197778.29;78.30 Doel78.31 et78.32 al18.104.22.168 199478.35;78.36 Kochneva-Pervukhova78.37 et78.38 al22.214.171.124 199878.41;78.42 Derkatch78.43 et78.44 al126.96.36.199 199978.47)188.8.131.52
Using both in vivo and in vitro assays, we established that PNM2-1 does not have a defect in aggregation or [PSI+] induction.
Using79.1 both79.2 in79.3 vivo79.4 and79.5 in79.6 vitro79.7 assays79.8,79.9 we79.10 established79.11 that79.12 PNM2-179.13 does79.14 not79.15 have79.16 a79.17 defect79.18 in79.19 aggregation79.20 or79.21 [79.22PSI79.23+79.24]79.25 induction184.108.40.206
Earlier work indicated that PNM2-1 is capable of seeding [PSI+] in vivo (Kochneva-Pervukhova et al. 1998; Derkatch et al. 1999; King 2001).
Earlier80.1 work80.2 indicated80.3 that80.4 PNM2-180.5 is80.6 capable80.7 of80.8 seeding80.9 [80.10PSI80.11+80.12]80.13 in80.14 vivo80.15 (80.16Kochneva-Pervukhova80.17 et80.18 al220.127.116.11 199880.21;80.22 Derkatch80.23 et80.24 al18.104.22.168 199980.27;80.28 King80.29 200180.30)22.214.171.124
Consistent with these reports, we found that overexpression of a PNM2-1-GFP fusion in [psi–] [PIN+] cells with a wild-type SUP35 locus led to both focus formation and [PSI+] induction (Figure 4A).
Consistent81.1 with81.2 these81.3 reports81.4,81.5 we81.6 found81.7 that81.8 overexpression81.9 of81.10 a81.11 PNM2-1-GFP81.12 fusion81.13 in81.14 [81.15psi81.16–81.17]81.18 [81.19PIN81.20+81.21]81.22 cells81.23 with81.24 a81.25 wild-type81.26 SUP3581.27 locus81.28 led81.29 to81.30 both81.31 focus81.32 formation81.33 and81.34 [81.35PSI81.36+81.37]81.38 induction81.39 (81.40Figure81.41 4A81.42)126.96.36.199
A previous study of Sup35p polymerization in extracts had suggested that PNM2-1 might interfere with [PSI+] through a defect in seeding (Kochneva-Pervukhova et al. 1998).
A82.1 previous82.2 study82.3 of82.4 Sup35p82.5 polymerization82.6 in82.7 extracts82.8 had82.9 suggested82.10 that82.11 PNM2-182.12 might82.13 interfere82.14 with82.15 [82.16PSI82.17+82.18]82.19 through82.20 a82.21 defect82.22 in82.23 seeding82.24 (82.25Kochneva-Pervukhova82.26 et82.27 al188.8.131.52 199882.30)184.108.40.206
We tested this by examining the rate of seeded polymerization of recombinant PNM2-1 protein.
We83.1 tested83.2 this83.3 by83.4 examining83.5 the83.6 rate83.7 of83.8 seeded83.9 polymerization83.10 of83.11 recombinant83.12 PNM2-183.13 protein220.127.116.11
Like wild-type Sup35p, purified PNM2-1 spontaneously formed amyloid fibrils in vitro; this was accelerated by the addition of preformed Sup35p polymer seeds (data not shown).
Like84.1 wild-type84.2 Sup35p84.3,84.4 purified84.5 PNM2-184.6 spontaneously84.7 formed84.8 amyloid84.9 fibrils84.10 in84.11 vitro84.12;84.13 this84.14 was84.15 accelerated84.16 by84.17 the84.18 addition84.19 of84.20 preformed84.21 Sup35p84.22 polymer84.23 seeds84.24 (84.25data84.26 not84.27 shown84.28)18.104.22.168
We measured the initial rates of polymerization of wild-type and PNM2-1 protein seeded by preformed wild-type polymers (Figure 4B) and by PNM2-1 polymers (Figure 4C) using a thioflavin-T–binding assay.
We85.1 measured85.2 the85.3 initial85.4 rates85.5 of85.6 polymerization85.7 of85.8 wild-type85.9 and85.10 PNM2-185.11 protein85.12 seeded85.13 by85.14 preformed85.15 wild-type85.16 polymers85.17 (85.18Figure85.19 4B85.20)85.21 and85.22 by85.23 PNM2-185.24 polymers85.25 (85.26Figure85.27 4C85.28)85.29 using85.30 a85.31 thioflavin-T85.32–85.33binding85.34 assay22.214.171.124
We observed that wild-type and PNM2-1 monomers were seeded by wild-type polymers with similar kinetics; likewise, PNM2-1 polymers seeded both wild-type and PNM2-1 monomers equivalently.
We86.1 observed86.2 that86.3 wild-type86.4 and86.5 PNM2-186.6 monomers86.7 were86.8 seeded86.9 by86.10 wild-type86.11 polymers86.12 with86.13 similar86.14 kinetics86.15;86.16 likewise86.17,86.18 PNM2-186.19 polymers86.20 seeded86.21 both86.22 wild-type86.23 and86.24 PNM2-186.25 monomers86.26 equivalently126.96.36.199
Thus, the PNM2-1 mutation does not affect polymerization or seeding.
Thus87.1,87.2 the87.3 PNM2-187.4 mutation87.5 does87.6 not87.7 affect87.8 polymerization87.9 or87.10 seeding188.8.131.52
Dissection of the Sup35p Prion Domain
Dissection89.1 of89.2 the89.3 Sup35p89.4 Prion89.5 Domain89.6
At top are schematic diagrams of these experiments; positive outcomes are shown below the arrows.
At90.1 top90.2 are90.3 schematic90.4 diagrams90.5 of90.6 these90.7 experiments90.8;90.9 positive90.10 outcomes90.11 are90.12 shown90.13 below90.14 the90.15 arrows184.108.40.206
In some cases, similar experiments have been reported by Parham et al. (2001) (indicated by “a”) and are repeated here as controls.
In91.1 some91.2 cases91.3,91.4 similar91.5 experiments91.6 have91.7 been91.8 reported91.9 by91.10 Parham91.11 et91.12 al220.127.116.11 (91.15200191.16)91.17 (91.18indicated91.19 by91.20 “91.21a91.22”91.23)91.24 and91.25 are91.26 repeated91.27 here91.28 as91.29 controls18.104.22.168
Plasmid-borne M-GFP fusions of the indicated Sup35p N domain fragments (green) were overexpressed in a [psi–] [PIN+] strain and examined for fluorescent focus formation.
Plasmid-borne93.1 M-GFP93.2 fusions93.3 of93.4 the93.5 indicated93.6 Sup35p93.7 N93.8 domain93.9 fragments93.10 (93.11green93.12)93.13 were93.14 overexpressed93.15 in93.16 a93.17 [93.18psi93.19–93.20]93.21 [93.22PIN93.23+93.24]93.25 strain93.26 and93.27 examined93.28 for93.29 fluorescent93.30 focus93.31 formation22.214.171.124
The symbol “+” indicates that 10% or more of cells displayed aggregates.
The94.1 symbol94.2 “94.3+94.4”94.5 indicates94.6 that94.7 10% or94.8 more94.9 of94.10 cells94.11 displayed94.12 aggregates126.96.36.199
Sup351–57-M-GFP displayed a lower frequency of aggregation (approximately 1%).
Sup35195.1–95.257-M-GFP95.3 displayed95.4 a95.5 lower95.6 frequency95.7 of95.8 aggregation95.9 (95.10approximately95.11 1%)188.8.131.52
Strains from the aggregation experiment were plated onto SD-ade medium and scored for growth to test whether aggregates of truncated protein (green) convert chromosomally encoded protein (blue) to [PSI+].
Strains97.1 from97.2 the97.3 aggregation97.4 experiment97.5 were97.6 plated97.7 onto97.8 SD-ade97.9 medium97.10 and97.11 scored97.12 for97.13 growth97.14 to97.15 test97.16 whether97.17 aggregates97.18 of97.19 truncated97.20 protein97.21 (97.22green97.23)97.24 convert97.25 chromosomally97.26 encoded97.27 protein97.28 (97.29blue97.30)97.31 to97.32 [97.33PSI97.34+97.35]184.108.40.206
The symbol “+” indicates approximately 5–10% conversion frequency.
The98.1 symbol98.2 “98.3+98.4”98.5 indicates98.6 approximately98.7 598.8–98.910% conversion98.10 frequency220.127.116.11
Consistent with the aggregation experiment, Sup351–57-M-GFP displayed a lower frequency of [PSI+] induction (approximately 1%).
Consistent99.1 with99.2 the99.3 aggregation99.4 experiment99.5,99.6 Sup35199.7–99.857-M-GFP99.9 displayed99.10 a99.11 lower99.12 frequency99.13 of99.14 [99.15PSI99.16+99.17]99.18 induction99.19 (99.20approximately99.21 1%)18.104.22.168
Indicated proteins were expressed as –M-GFP fusions in [PSI+] [PIN+] cells, which were examined to determine whether GFP-labeled truncations (green) decorate preexisting aggregates of full-length Sup35p (blue).
Indicated101.1 proteins101.2 were101.3 expressed101.4 as101.5 –101.6M-GFP101.7 fusions101.8 in101.9 [101.10PSI101.11+101.12]101.13 [101.14PIN101.15+101.16]101.17 cells101.18,101.19 which101.20 were101.21 examined101.22 to101.23 determine101.24 whether101.25 GFP-labeled101.26 truncations101.27 (101.28green101.29)101.30 decorate101.31 preexisting101.32 aggregates101.33 of101.34 full-length101.35 Sup35p101.36 (101.37blue101.38)22.214.171.124
Curiously, Sup351–49-M-GFP in [PSI+] cells formed abnormally large “ribbon” aggregates of the kind typically observed during de novo [PSI+] induction; furthermore, approximately 10% of the cells reverted to [psi–] (indicated by “*”).
Curiously102.1,102.2 Sup351102.3–102.449-M-GFP102.5 in102.6 [102.7PSI102.8+102.9]102.10 cells102.11 formed102.12 abnormally102.13 large102.14 “102.15ribbon102.16”102.17 aggregates102.18 of102.19 the102.20 kind102.21 typically102.22 observed102.23 during102.24 de102.25 novo102.26 [102.27PSI102.28+102.29]102.30 induction102.31;102.32 furthermore102.33,102.34 approximately102.35 10% of102.36 the102.37 cells102.38 reverted102.39 to102.40 [102.41psi102.42–102.43]102.44 (102.45indicated102.46 by102.47 “102.48*102.49”102.50)126.96.36.199
Thus, this truncation was a potent dominant PNM mutant.
Thus103.1,103.2 this103.3 truncation103.4 was103.5 a103.6 potent103.7 dominant103.8 PNM103.9 mutant188.8.131.52
A SUP35-deleted [PSI+] [PIN+] bearing wild-type SUP35 maintainer (blue) was transformed with maintainer plasmids containing the indicated truncation (purple).
A105.1 SUP35-deleted105.2 [105.3PSI105.4+105.5]105.6 [105.7PIN105.8+105.9]105.10 bearing105.11 wild-type105.12 SUP35105.13 maintainer105.14 (105.15blue105.16)105.17 was105.18 transformed105.19 with105.20 maintainer105.21 plasmids105.22 containing105.23 the105.24 indicated105.25 truncation105.26 (105.27purple105.28)184.108.40.206
The wild-type maintainer was lost by counterselection, and the resulting strain was tested for [PSI+] by color and growth on SD-ade medium.
The106.1 wild-type106.2 maintainer106.3 was106.4 lost106.5 by106.6 counterselection106.7,106.8 and106.9 the106.10 resulting106.11 strain106.12 was106.13 tested106.14 for106.15 [106.16PSI106.17+106.18]106.19 by106.20 color106.21 and106.22 growth106.23 on106.24 SD-ade106.25 medium220.127.116.11
The Sup351–93 mutant displayed an intermediate pink color and grew poorly on SD-ade medium, as previously reported (Parham et al. 2001).
The107.1 Sup351107.2–107.393107.4 mutant107.5 displayed107.6 an107.7 intermediate107.8 pink107.9 color107.10 and107.11 grew107.12 poorly107.13 on107.14 SD-ade107.15 medium107.16,107.17 as107.18 previously107.19 reported107.20 (107.21Parham107.22 et107.23 al18.104.22.168 2001107.26)22.214.171.124
King (2001) reports that Sup351–61-GFP fusion could decorate [PSI+] aggregates in certain strains and could induce [PSI+] de novo when overexpressed.
King109.1 (109.22001109.3)109.4 reports109.5 that109.6 Sup351109.7–109.861-GFP109.9 fusion109.10 could109.11 decorate109.12 [109.13PSI109.14+109.15]109.16 aggregates109.17 in109.18 certain109.19 strains109.20 and109.21 could109.22 induce109.23 [109.24PSI109.25+109.26]109.27 de109.28 novo109.29 when109.30 overexpressed126.96.36.199
Instead, the PNM2-1 strain shows a marked defect in the inheritance of [PSI+].
Instead110.1,110.2 the110.3 PNM2-1110.4 strain110.5 shows110.6 a110.7 marked110.8 defect110.9 in110.10 the110.11 inheritance110.12 of110.13 [110.14PSI110.15+110.16]188.8.131.52
When the wild-type SUP35 gene of a [PSI+] strain was replaced with PNM2-1, the strain retained the prion on synthetic defined (SD) yeast medium that selected for [PSI+] (SD-ade medium) but reverted to [psi–] at a high frequency in nonselective YEPD medium, resulting in sectored colonies (Figure 4D).
When111.1 the111.2 wild-type111.3 SUP35111.4 gene111.5 of111.6 a111.7 [111.8PSI111.9+111.10]111.11 strain111.12 was111.13 replaced111.14 with111.15 PNM2-1111.16,111.17 the111.18 strain111.19 retained111.20 the111.21 prion111.22 on111.23 synthetic111.24 defined111.25 (111.26SD111.27)111.28 yeast111.29 medium111.30 that111.31 selected111.32 for111.33 [111.34PSI111.35+111.36]111.37 (111.38SD-ade111.39 medium111.40)111.41 but111.42 reverted111.43 to111.44 [111.45psi111.46–111.47]111.48 at111.49 a111.50 high111.51 frequency111.52 in111.53 nonselective111.54 YEPD111.55 medium111.56,111.57 resulting111.58 in111.59 sectored111.60 colonies111.61 (111.62Figure111.63 4D111.64)184.108.40.206
We measured the rate of [PSI+] loss in a PNM2-1 strain by growing it in YEPD medium and, at various time points, plating aliquots of the culture onto SD-ade medium to determine the fraction of cells that had retained [PSI+] (Figure 4E).
We112.1 measured112.2 the112.3 rate112.4 of112.5 [112.6PSI112.7+112.8]112.9 loss112.10 in112.11 a112.12 PNM2-1112.13 strain112.14 by112.15 growing112.16 it112.17 in112.18 YEPD112.19 medium112.20 and112.21,112.22 at112.23 various112.24 time112.25 points112.26,112.27 plating112.28 aliquots112.29 of112.30 the112.31 culture112.32 onto112.33 SD-ade112.34 medium112.35 to112.36 determine112.37 the112.38 fraction112.39 of112.40 cells112.41 that112.42 had112.43 retained112.44 [112.45PSI112.46+112.47]112.48 (112.49Figure112.50 4E112.51)220.127.116.11
A wild-type strain retained [PSI+] in all of the cells throughout the experiment.
A113.1 wild-type113.2 strain113.3 retained113.4 [113.5PSI113.6+113.7]113.8 in113.9 all113.10 of113.11 the113.12 cells113.13 throughout113.14 the113.15 experiment18.104.22.168
By contrast, in the PNM2-1 strain the fraction of [PSI+] cells decreased rapidly while the cells grew logarithmically, but remained at a constant level when the cells entered stationary phase.
By114.1 contrast114.2,114.3 in114.4 the114.5 PNM2-1114.6 strain114.7 the114.8 fraction114.9 of114.10 [114.11PSI114.12+114.13]114.14 cells114.15 decreased114.16 rapidly114.17 while114.18 the114.19 cells114.20 grew114.21 logarithmically114.22,114.23 but114.24 remained114.25 at114.26 a114.27 constant114.28 level114.29 when114.30 the114.31 cells114.32 entered114.33 stationary114.34 phase22.214.171.124
These findings indicate that PNM2-1 acts to eliminate [PSI+] in dividing cells, consistent with a defect in prion replication.
These115.1 findings115.2 indicate115.3 that115.4 PNM2-1115.5 acts115.6 to115.7 eliminate115.8 [115.9PSI115.10+115.11]115.12 in115.13 dividing115.14 cells115.15,115.16 consistent115.17 with115.18 a115.19 defect115.20 in115.21 prion115.22 replication126.96.36.199
We next used a recently described assay to measure the number of heritable prion seeds (propagons) in a PNM2-1 strain.
We116.1 next116.2 used116.3 a116.4 recently116.5 described116.6 assay116.7 to116.8 measure116.9 the116.10 number116.11 of116.12 heritable116.13 prion116.14 seeds116.15 (116.16propagons116.17)116.18 in116.19 a116.20 PNM2-1116.21 strain188.8.131.52
Here, prion replication is inhibited by GuHCl treatment.
Here117.1,117.2 prion117.3 replication117.4 is117.5 inhibited117.6 by117.7 GuHCl117.8 treatment184.108.40.206
As the cells divide, preexisting propagons are diluted but not destroyed.
As118.1 the118.2 cells118.3 divide118.4,118.5 preexisting118.6 propagons118.7 are118.8 diluted118.9 but118.10 not118.11 destroyed220.127.116.11
The number of propagons present in a colony arising from a single cell is then evaluated by removing the GuHCl prion replication block after a large number (10 or more) of cell divisions and counting the total number of [PSI+] cells in that colony (Cox et al. 2003).
The119.1 number119.2 of119.3 propagons119.4 present119.5 in119.6 a119.7 colony119.8 arising119.9 from119.10 a119.11 single119.12 cell119.13 is119.14 then119.15 evaluated119.16 by119.17 removing119.18 the119.19 GuHCl119.20 prion119.21 replication119.22 block119.23 after119.24 a119.25 large119.26 number119.27 (119.2810119.29 or119.30 more119.31)119.32 of119.33 cell119.34 divisions119.35 and119.36 counting119.37 the119.38 total119.39 number119.40 of119.41 [119.42PSI119.43+119.44]119.45 cells119.46 in119.47 that119.48 colony119.49 (119.50Cox119.51 et119.52 al18.104.22.168 2003119.55)22.214.171.124
Whereas a wild-type strain had a median of 92 (n = 24) propagons per cell, the PNM2-1 strain had dramatically fewer: 41 of 50 cells had no [PSI+] propagons at all (i.e., were [psi–]), and among the remaining nine [PSI+] cells, the median propagon number was six (Figure 4F).
Whereas120.1 a120.2 wild-type120.3 strain120.4 had120.5 a120.6 median120.7 of120.8 92120.9 (120.10n120.11 = 24120.12)120.13 propagons120.14 per120.15 cell120.16,120.17 the120.18 PNM2-1120.19 strain120.20 had120.21 dramatically120.22 fewer120.23:120.24 41120.25 of120.26 50120.27 cells120.28 had120.29 no120.30 [120.31PSI120.32+120.33]120.34 propagons120.35 at120.36 all120.37 (120.38i126.96.36.199e188.8.131.52,120.43 were120.44 [120.45psi120.46–120.47]120.48)120.49,120.50 and120.51 among120.52 the120.53 remaining120.54 nine120.55 [120.56PSI120.57+120.58]120.59 cells120.60,120.61 the120.62 median120.63 propagon120.64 number120.65 was120.66 six120.67 (120.68Figure120.69 4F120.70)184.108.40.206
Thus, although a PNM2-1 strain can harbor [PSI+] prions, a defect in propagon replication causes mitotic instability, demonstrating the importance of oligopeptide repeat 2 in prion replication or segregation.
Thus121.1,121.2 although121.3 a121.4 PNM2-1121.5 strain121.6 can121.7 harbor121.8 [121.9PSI121.10+121.11]121.12 prions121.13,121.14 a121.15 defect121.16 in121.17 propagon121.18 replication121.19 causes121.20 mitotic121.21 instability121.22,121.23 demonstrating121.24 the121.25 importance121.26 of121.27 oligopeptide121.28 repeat121.29 2121.30 in121.31 prion121.32 replication121.33 or121.34 segregation220.127.116.11
PNM2–1 (G58D) Prevents Inheritance But Not Aggregation of Sup35p Prions
PNM2123.1–123.21123.3 (123.4G58D123.5)123.6 Prevents123.7 Inheritance123.8 But123.9 Not123.10 Aggregation123.11 of123.12 Sup35p123.13 Prions123.14
(A) PNM2-1 protein can seed [PSI+].
(124.1A124.2)124.3 PNM2-1124.4 protein124.5 can124.6 seed124.7 [124.8PSI124.9+124.10]18.104.22.168
A Sup35p inducer containing the PNM2-1 (G58D) mutation was overexpressed in [psi–] [PIN+] cells; shown are cells (inset) with representative fluorescent foci, which were the same in frequency and appearance as cells with a wild-type inducer.
A125.1 Sup35p125.2 inducer125.3 containing125.4 the125.5 PNM2-1125.6 (125.7G58D125.8)125.9 mutation125.10 was125.11 overexpressed125.12 in125.13 [125.14psi125.15–125.16]125.17 [125.18PIN125.19+125.20]125.21 cells125.22;125.23 shown125.24 are125.25 cells125.26 (125.27inset125.28)125.29 with125.30 representative125.31 fluorescent125.32 foci125.33,125.34 which125.35 were125.36 the125.37 same125.38 in125.39 frequency125.40 and125.41 appearance125.42 as125.43 cells125.44 with125.45 a125.46 wild-type125.47 inducer22.214.171.124
Cells overexpressing inducer versions of wild-type Sup35p (SUP), an aggregation-defective N-terminal truncation (Δ1–38), and PNM2-1 were plated and scored for Ade+.
Cells126.1 overexpressing126.2 inducer126.3 versions126.4 of126.5 wild-type126.6 Sup35p126.7 (126.8SUP126.9)126.10,126.11 an126.12 aggregation-defective126.13 N-terminal126.14 truncation126.15 (126.16Δ1126.17–126.1838126.19)126.20,126.21 and126.22 PNM2-1126.23 were126.24 plated126.25 and126.26 scored126.27 for126.28 Ade126.29+126.96.36.199
Approximately 1000 colonies were counted.
Approximately127.1 1000127.2 colonies127.3 were127.4 counted127.5.127.6
(B) PNM2-1 protein polymerization is similar to that of wild-type protein.
(128.1B128.2)128.3 PNM2-1128.4 protein128.5 polymerization128.6 is128.7 similar128.8 to128.9 that128.10 of128.11 wild-type128.12 protein188.8.131.52
(C) Preformed PNM2-1 polymers seed wild-type and PNM2-1 monomers with comparable efficiency.
(129.1C129.2)129.3 Preformed129.4 PNM2-1129.5 polymers129.6 seed129.7 wild-type129.8 and129.9 PNM2-1129.10 monomers129.11 with129.12 comparable129.13 efficiency184.108.40.206
Endpoint PNM2-1 polymers were used to seed fresh reactions.
Endpoint130.1 PNM2-1130.2 polymers130.3 were130.4 used130.5 to130.6 seed130.7 fresh130.8 reactions220.127.116.11
(D) PNM2-1 displays a partially dominant, incompletely penetrant defect in [PSI+] maintenance.
(131.1D131.2)131.3 PNM2-1131.4 displays131.5 a131.6 partially131.7 dominant131.8,131.9 incompletely131.10 penetrant131.11 defect131.12 in131.13 [131.14PSI131.15+131.16]131.17 maintenance18.104.22.168
[psi–] (1) and [PSI+] (2) SUP35::TRP1 pSUP35 controls are shown.
[132.1psi132.2–132.3]132.4 (132.51132.6)132.7 and132.8 [132.9PSI132.10+132.11]132.12 (132.132132.14)132.15 SUP35132.16:132.17:132.18TRP1132.19 pSUP35132.20 controls132.21 are132.22 shown22.214.171.124
[PSI+] [PIN+] SUP35::TRP1 pSUP35 was transformed with a second maintainer expressing PNM2-1 (3).
[133.1PSI133.2+133.3]133.4 [133.5PIN133.6+133.7]133.8 SUP35133.9:133.10:133.11TRP1133.12 pSUP35133.13 was133.14 transformed133.15 with133.16 a133.17 second133.18 maintainer133.19 expressing133.20 PNM2-1133.21 (133.223133.23)126.96.36.199
The wild-type maintainer (pSUP35) was then lost through counterselection (4).
The134.1 wild-type134.2 maintainer134.3 (134.4pSUP35134.5)134.6 was134.7 then134.8 lost134.9 through134.10 counterselection134.11 (134.124134.13)188.8.131.52
Red sectors from (4) were isolated, retransformed with the wild-type maintainer, and allowed to lose the PNM2-1 maintainer (5).
Red135.1 sectors135.2 from135.3 (135.44135.5)135.6 were135.7 isolated135.8,135.9 retransformed135.10 with135.11 the135.12 wild-type135.13 maintainer135.14,135.15 and135.16 allowed135.17 to135.18 lose135.19 the135.20 PNM2-1135.21 maintainer135.22 (135.235135.24)184.108.40.206
(E) Mitotic instability of [PSI+] in the PNM2-1 strain.
(136.1E136.2)136.3 Mitotic136.4 instability136.5 of136.6 [136.7PSI136.8+136.9]136.10 in136.11 the136.12 PNM2-1136.13 strain220.127.116.11
A pink (Ade+) [PSI+] [PIN+] PNM2-1 isolate was grown to log phase in SD-ade liquid then shifted into nonselective (YEPD) medium.
A137.1 pink137.2 (137.3Ade137.4+137.5)137.6 [137.7PSI137.8+137.9]137.10 [137.11PIN137.12+137.13]137.14 PNM2-1137.15 isolate137.16 was137.17 grown137.18 to137.19 log137.20 phase137.21 in137.22 SD-ade137.23 liquid137.24 then137.25 shifted137.26 into137.27 nonselective137.28 (137.29YEPD137.30)137.31 medium18.104.22.168
At indicated time points, aliquots were plated onto SD-ade and YEPD media to determine the fraction of [PSI+] cells (minimum of 200 colonies counted per time point).
At138.1 indicated138.2 time138.3 points138.4,138.5 aliquots138.6 were138.7 plated138.8 onto138.9 SD-ade138.10 and138.11 YEPD138.12 media138.13 to138.14 determine138.15 the138.16 fraction138.17 of138.18 [138.19PSI138.20+138.21]138.22 cells138.23 (138.24minimum138.25 of138.26 200138.27 colonies138.28 counted138.29 per138.30 time138.31 point138.32)22.214.171.124
Whereas a wild-type control remained [PSI+] through the experiment, the PNM2-1 strain rapidly lost [PSI+] during logarithmic growth; during stationary phase (18 h and beyond), the percentage of [PSI+] PNM2-1 strains remained unchanged (approximately 5%).
Whereas139.1 a139.2 wild-type139.3 control139.4 remained139.5 [139.6PSI139.7+139.8]139.9 through139.10 the139.11 experiment139.12,139.13 the139.14 PNM2-1139.15 strain139.16 rapidly139.17 lost139.18 [139.19PSI139.20+139.21]139.22 during139.23 logarithmic139.24 growth139.25;139.26 during139.27 stationary139.28 phase139.29 (139.3018139.31 h139.32 and139.33 beyond139.34)139.35,139.36 the139.37 percentage139.38 of139.39 [139.40PSI139.41+139.42]139.43 PNM2-1139.44 strains139.45 remained139.46 unchanged139.47 (139.48approximately139.49 5%)126.96.36.199
(F) Propagon count of PNM2-1 vs. wild-type [PSI+] strains.
(140.1F140.2)140.3 Propagon140.4 count140.5 of140.6 PNM2-1140.7 vs188.8.131.52 wild-type140.10 [140.11PSI140.12+140.13]140.14 strains184.108.40.206
The majority of PNM2-1 cells had no [PSI+] propagons (i.e., were [psi–]).
The141.1 majority141.2 of141.3 PNM2-1141.4 cells141.5 had141.6 no141.7 [141.8PSI141.9+141.10]141.11 propagons141.12 (141.13i220.127.116.11e18.104.22.168,141.18 were141.19 [141.20psi141.21–141.22]141.23)22.214.171.124
In both strains, a small number of “jackpot” cells contained over 200 propagons; see Cox et al. (2003).
In142.1 both142.2 strains142.3,142.4 a142.5 small142.6 number142.7 of142.8 “142.9jackpot142.10”142.11 cells142.12 contained142.13 over142.14 200142.15 propagons142.16;142.17 see142.18 Cox142.19 et142.20 al126.96.36.199 (142.232003142.24)188.8.131.52
Design of Novel Prion Domains
Design143.1 of143.2 Novel143.3 Prion143.4 Domains143.5
Our data suggested that the formation and inheritance of prions involve distinct regions of Sup35p and New1p prion domains.
Our144.1 data144.2 suggested144.3 that144.4 the144.5 formation144.6 and144.7 inheritance144.8 of144.9 prions144.10 involve144.11 distinct144.12 regions144.13 of144.14 Sup35p144.15 and144.16 New1p144.17 prion144.18 domains184.108.40.206
To assess the interchangeability of these prion domain components, we constructed a chimeric prion domain, termed F, in which the aggregation-determining NYN repeat of New1p was fused to the oligopeptide repeats of Sup35p (Figure 5A).
To145.1 assess145.2 the145.3 interchangeability145.4 of145.5 these145.6 prion145.7 domain145.8 components145.9,145.10 we145.11 constructed145.12 a145.13 chimeric145.14 prion145.15 domain145.16,145.17 termed145.18 F145.19,145.20 in145.21 which145.22 the145.23 aggregation-determining145.24 NYN145.25 repeat145.26 of145.27 New1p145.28 was145.29 fused145.30 to145.31 the145.32 oligopeptide145.33 repeats145.34 of145.35 Sup35p145.36 (145.37Figure145.38 5A145.39)220.127.116.11
While initially soluble and active, a fusion of F and the Sup35p M and C domains (F-M-C) could be converted into an aggregated state, termed [F+], after transient overexpression of F-M-GFP.
While146.1 initially146.2 soluble146.3 and146.4 active146.5,146.6 a146.7 fusion146.8 of146.9 F146.10 and146.11 the146.12 Sup35p146.13 M146.14 and146.15 C146.16 domains146.17 (146.18F-M-C146.19)146.20 could146.21 be146.22 converted146.23 into146.24 an146.25 aggregated146.26 state146.27,146.28 termed146.29 [146.30F146.31+146.32]146.33,146.34 after146.35 transient146.36 overexpression146.37 of146.38 F-M-GFP18.104.22.168
As with [NU+], [F+] induction did not require [PIN+] (data not shown).
As147.1 with147.2 [147.3NU147.4+147.5]147.6,147.7 [147.8F147.9+147.10]147.11 induction147.12 did147.13 not147.14 require147.15 [147.16PIN147.17+147.18]147.19 (147.20data147.21 not147.22 shown147.23)22.214.171.124
[F+] could be eliminated by GuHCl treatment (Figure 5B) and was inherited in a dominant, non-Mendelian manner (Figure 5C).
[148.1F148.2+148.3]148.4 could148.5 be148.6 eliminated148.7 by148.8 GuHCl148.9 treatment148.10 (148.11Figure148.12 5B148.13)148.14 and148.15 was148.16 inherited148.17 in148.18 a148.19 dominant148.20,148.21 non-Mendelian148.22 manner148.23 (148.24Figure148.25 5C148.26)126.96.36.199
As with Sup35p in a [PSI+] strain, F-M-C protein in [F+] but not in [f –] extracts sedimented entirely to the pellet fraction following high-speed centrifugation (Figure 5D).
As149.1 with149.2 Sup35p149.3 in149.4 a149.5 [149.6PSI149.7+149.8]149.9 strain149.10,149.11 F-M-C149.12 protein149.13 in149.14 [149.15F149.16+149.17]149.18 but149.19 not149.20 in149.21 [149.22f149.23 –149.24]149.25 extracts149.26 sedimented149.27 entirely149.28 to149.29 the149.30 pellet149.31 fraction149.32 following149.33 high-speed149.34 centrifugation149.35 (149.36Figure149.37 5D149.38)188.8.131.52
Thus, [F+] results from a prion state of F-M-C.
Thus150.1,150.2 [150.3F150.4+150.5]150.6 results150.7 from150.8 a150.9 prion150.10 state150.11 of150.12 F-M-C184.108.40.206
We next explored the specificity of [F+] prion seeding.
We151.1 next151.2 explored151.3 the151.4 specificity151.5 of151.6 [151.7F151.8+151.9]151.10 prion151.11 seeding220.127.116.11
Overexpression of the Sup35p prion domain did not induce [F+]; conversely, F-M-GFP overexpression did not induce [PSI+] (Figure 5E).
Overexpression152.1 of152.2 the152.3 Sup35p152.4 prion152.5 domain152.6 did152.7 not152.8 induce152.9 [152.10F152.11+152.12]152.13;152.14 conversely152.15,152.16 F-M-GFP152.17 overexpression152.18 did152.19 not152.20 induce152.21 [152.22PSI152.23+152.24]152.25 (152.26Figure152.27 5E152.28)18.104.22.168
However, F-M-GFP readily induced [NU+], indicating that mismatched sequences outside of the aggregating region did not prevent cross-interactions between heterologous proteins.
However153.1,153.2 F-M-GFP153.3 readily153.4 induced153.5 [153.6NU153.7+153.8]153.9,153.10 indicating153.11 that153.12 mismatched153.13 sequences153.14 outside153.15 of153.16 the153.17 aggregating153.18 region153.19 did153.20 not153.21 prevent153.22 cross-interactions153.23 between153.24 heterologous153.25 proteins22.214.171.124
Interestingly, overexpression of New11–53-GFP induced Ade+ colonies in the [f –] strain, but this adenine prototrophy proved unstable.
Interestingly154.1,154.2 overexpression154.3 of154.4 New11154.5–154.653-GFP154.7 induced154.8 Ade154.9+154.10 colonies154.11 in154.12 the154.13 [154.14f154.15 –154.16]154.17 strain154.18,154.19 but154.20 this154.21 adenine154.22 prototrophy154.23 proved154.24 unstable126.96.36.199
We also examined the ability of preexisting prion aggregates to recruit different prion-forming proteins using an antisuppression assay (Santoso et al. 2000) (Figure 5F).
We155.1 also155.2 examined155.3 the155.4 ability155.5 of155.6 preexisting155.7 prion155.8 aggregates155.9 to155.10 recruit155.11 different155.12 prion-forming155.13 proteins155.14 using155.15 an155.16 antisuppression155.17 assay155.18 (155.19Santoso155.20 et155.21 al188.8.131.52 2000155.24)155.25 (155.26Figure155.27 5F155.28)184.108.40.206
[PSI+], [F+], and [NU+] strains were transformed with Sup35p–, F-M-C– or New11–153-M-C–encoding plasmids; the color of the resulting colonies indicates whether the second maintainer protein is soluble (red) or aggregates as a result of the resident prion (pink/white).
[156.1PSI156.2+156.3]156.4,156.5 [156.6F156.7+156.8]156.9,156.10 and156.11 [156.12NU156.13+156.14]156.15 strains156.16 were156.17 transformed156.18 with156.19 Sup35p156.20–156.21,156.22 F-M-C156.23–156.24 or156.25 New11156.26–156.27153-M-C156.28–156.29encoding156.30 plasmids156.31;156.32 the156.33 color156.34 of156.35 the156.36 resulting156.37 colonies156.38 indicates156.39 whether156.40 the156.41 second156.42 maintainer156.43 protein156.44 is156.45 soluble156.46 (156.47red156.48)156.49 or156.50 aggregates156.51 as156.52 a156.53 result156.54 of156.55 the156.56 resident156.57 prion156.58 (156.59pink/white156.60)220.127.116.11
Consistent with the induction data, F-M-C and New11–153-M-C were not incorporated into [PSI+] aggregates; likewise, Sup35p did not interact with [F+] or [NU+] aggregates.
Consistent157.1 with157.2 the157.3 induction157.4 data157.5,157.6 F-M-C157.7 and157.8 New11157.9–157.10153-M-C157.11 were157.12 not157.13 incorporated157.14 into157.15 [157.16PSI157.17+157.18]157.19 aggregates157.20;157.21 likewise157.22,157.23 Sup35p157.24 did157.25 not157.26 interact157.27 with157.28 [157.29F157.30+157.31]157.32 or157.33 [157.34NU157.35+157.36]157.37 aggregates18.104.22.168
However, [F+] prions recruited New11–153-M-C and, to a lesser extent, [NU+] recruited F-M-C.
However158.1,158.2 [158.3F158.4+158.5]158.6 prions158.7 recruited158.8 New11158.9–158.10153-M-C158.11 and158.12,158.13 to158.14 a158.15 lesser158.16 extent158.17,158.18 [158.19NU158.20+158.21]158.22 recruited158.23 F-M-C22.214.171.124
Thus, F and New1p prion domains can cross-interact during de novo induction and at normal levels of expression, indicating that the NYN repeat is sufficient to specify homotypic interaction between two otherwise distinct prion domains.
Thus159.1,159.2 F159.3 and159.4 New1p159.5 prion159.6 domains159.7 can159.8 cross-interact159.9 during159.10 de159.11 novo159.12 induction159.13 and159.14 at159.15 normal159.16 levels159.17 of159.18 expression159.19,159.20 indicating159.21 that159.22 the159.23 NYN159.24 repeat159.25 is159.26 sufficient159.27 to159.28 specify159.29 homotypic159.30 interaction159.31 between159.32 two159.33 otherwise159.34 distinct159.35 prion159.36 domains126.96.36.199
F, A New1p–Sup35p Chimera, Shows Prion Characteristics of New1p
F161.1,161.2 A161.3 New1p161.4–161.5Sup35p161.6 Chimera161.7,161.8 Shows161.9 Prion161.10 Characteristics161.11 of161.12 New1p161.13
(A) Schematic diagram illustrating the construction of chimera F.
(162.1A162.2)162.3 Schematic162.4 diagram162.5 illustrating162.6 the162.7 construction162.8 of162.9 chimera162.10 F188.8.131.52
(B) Chimera F forms a prion, [F+].
(163.1B163.2)163.3 Chimera163.4 F163.5 forms163.6 a163.7 prion163.8,163.9 [163.10F163.11+163.12]184.108.40.206
The SUP35 gene in a [psi–] [pin–] strain was replaced with the F-M-C fusion; after transient overexpression of F-M-GFP, approximately 10% of these cells converted from an Ade- ([f –]) to an Ade+ ([F+]) state.
The164.1 SUP35164.2 gene164.3 in164.4 a164.5 [164.6psi164.7–164.8]164.9 [164.10pin164.11–164.12]164.13 strain164.14 was164.15 replaced164.16 with164.17 the164.18 F-M-C164.19 fusion164.20;164.21 after164.22 transient164.23 overexpression164.24 of164.25 F-M-GFP164.26,164.27 approximately164.28 10% of164.29 these164.30 cells164.31 converted164.32 from164.33 an164.34 Ade- (164.35[164.36f164.37 –164.38]164.39)164.40 to164.41 an164.42 Ade164.43+164.44 (164.45[164.46F164.47+164.48]164.49)164.50 state220.127.116.11
Shown are examples of[f –] and [F+] strains, before and after GuHCl treatment, along with [psi–] and [PSI+] controls.
Shown165.1 are165.2 examples165.3 of165.4[165.5f165.6 –165.7]165.8 and165.9 [165.10F165.11+165.12]165.13 strains165.14,165.15 before165.16 and165.17 after165.18 GuHCl165.19 treatment165.20,165.21 along165.22 with165.23 [165.24psi165.25–165.26]165.27 and165.28 [165.29PSI165.30+165.31]165.32 controls18.104.22.168
(C) Non-Mendelian inheritance of [F+].
(166.1C166.2)166.3 Non-Mendelian166.4 inheritance166.5 of166.6 [166.7F166.8+166.9]22.214.171.124
A diploid made by mating a [F+] MATα strain against an [f –] MATα displayed a [F+] phenotype and, when sporulated, produced four [F+] meiotic progeny.
A167.1 diploid167.2 made167.3 by167.4 mating167.5 a167.6 [167.7F167.8+167.9]167.10 MATα167.11 strain167.12 against167.13 an167.14 [167.15f167.16 –167.17]167.18 MATα167.19 displayed167.20 a167.21 [167.22F167.23+167.24]167.25 phenotype167.26 and167.27,167.28 when167.29 sporulated167.30,167.31 produced167.32 four167.33 [167.34F167.35+167.36]167.37 meiotic167.38 progeny126.96.36.199
All 11 tetrads examined showed this 4:0 pattern of inheritance.
All168.1 11168.2 tetrads168.3 examined168.4 showed168.5 this168.6 4168.7:168.80168.9 pattern168.10 of168.11 inheritance188.8.131.52
(D) Sedimentation analysis of F-M-C.
(169.1D169.2)169.3 Sedimentation169.4 analysis169.5 of169.6 F-M-C184.108.40.206
Extracts of [f –] and [F+] strains, along with [psi–] and [PSI+] controls, were subjected to 50K × g ultracentrifugation for 15 min.
Extracts170.1 of170.2 [170.3f170.4 –170.5]170.6 and170.7 [170.8F170.9+170.10]170.11 strains170.12,170.13 along170.14 with170.15 [170.16psi170.17–170.18]170.19 and170.20 [170.21PSI170.22+170.23]170.24 controls170.25,170.26 were170.27 subjected170.28 to170.29 50K170.30 × g170.31 ultracentrifugation170.32 for170.33 15170.34 min220.127.116.11
Total, supernatant, and pellet fractions were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-SUP35NM serum.
Total171.1,171.2 supernatant171.3,171.4 and171.5 pellet171.6 fractions171.7 were171.8 separated171.9 by171.10 SDS-PAGE171.11,171.12 transferred171.13 to171.14 nitrocellulose171.15,171.16 and171.17 probed171.18 with171.19 anti-SUP35NM171.20 serum18.104.22.168
As with Sup35p, the prion form of F-M-C sediments primarily to the pellet but remains in the supernatant in [f –].
As172.1 with172.2 Sup35p172.3,172.4 the172.5 prion172.6 form172.7 of172.8 F-M-C172.9 sediments172.10 primarily172.11 to172.12 the172.13 pellet172.14 but172.15 remains172.16 in172.17 the172.18 supernatant172.19 in172.20 [172.21f172.22 –172.23]172.24.172.25
(E) F-M-GFP overexpression induces [NU+] but not [PSI+].
(173.1E173.2)173.3 F-M-GFP173.4 overexpression173.5 induces173.6 [173.7NU173.8+173.9]173.10 but173.11 not173.12 [173.13PSI173.14+173.15]22.214.171.124
Indicated inducers and maintainers were used in an induction experiment.
Indicated174.1 inducers174.2 and174.3 maintainers174.4 were174.5 used174.6 in174.7 an174.8 induction174.9 experiment126.96.36.199
The symbol “+” indicates approximately 5–10% conversion to Ade+.
The175.1 symbol175.2 “175.3+175.4”175.5 indicates175.6 approximately175.7 5175.8–175.910% conversion175.10 to175.11 Ade175.12+188.8.131.52
F induced [NU+] at a comparable efficiency to New11–153; although New11–153 overexpression promoted the appearance of Ade+ colonies in the F-M-C strain, these were fewer in number (less than 5%) and reverted to Ade- after restreaking.
F176.1 induced176.2 [176.3NU176.4+176.5]176.6 at176.7 a176.8 comparable176.9 efficiency176.10 to176.11 New11176.12–176.13153176.14;176.15 although176.16 New11176.17–176.18153176.19 overexpression176.20 promoted176.21 the176.22 appearance176.23 of176.24 Ade176.25+176.26 colonies176.27 in176.28 the176.29 F-M-C176.30 strain176.31,176.32 these176.33 were176.34 fewer176.35 in176.36 number176.37 (176.38less176.39 than176.40 5%)176.41 and176.42 reverted176.43 to176.44 Ade- after176.45 restreaking184.108.40.206
(F) [F+] and [NU+] prion proteins interact with each other but not with [PSI+].
(177.1F177.2)177.3 [177.4F177.5+177.6]177.7 and177.8 [177.9NU177.10+177.11]177.12 prion177.13 proteins177.14 interact177.15 with177.16 each177.17 other177.18 but177.19 not177.20 with177.21 [177.22PSI177.23+177.24]220.127.116.11
Episomal “second maintainers” were introduced into the indicated strains, along with an empty vector control.
Episomal178.1 “178.2second178.3 maintainers178.4”178.5 were178.6 introduced178.7 into178.8 the178.9 indicated178.10 strains178.11,178.12 along178.13 with178.14 an178.15 empty178.16 vector178.17 control18.104.22.168
Antisuppression (red) indicates that the second maintainer is soluble, while white/pink indicates coaggregation of the endogenous and episomal maintainers.
Antisuppression179.1 (179.2red179.3)179.4 indicates179.5 that179.6 the179.7 second179.8 maintainer179.9 is179.10 soluble179.11,179.12 while179.13 white/pink179.14 indicates179.15 coaggregation179.16 of179.17 the179.18 endogenous179.19 and179.20 episomal179.21 maintainers22.214.171.124
Can a simple aggregation-prone sequence such as polyglutamine (Zoghbi and Orr 2000) be turned into a heritable prion by fusion to an oligopeptide repeat?
Can180.1 a180.2 simple180.3 aggregation-prone180.4 sequence180.5 such180.6 as180.7 polyglutamine180.8 (180.9Zoghbi180.10 and180.11 Orr180.12 2000180.13)180.14 be180.15 turned180.16 into180.17 a180.18 heritable180.19 prion180.20 by180.21 fusion180.22 to180.23 an180.24 oligopeptide180.25 repeat?
We designed artificial prion domains containing short (Q22) and pathogenically expanded (Q62) polyglutamine tracts, either alone or adjacent to the Sup35p oligopeptide repeat (Figure 6A).
We181.1 designed181.2 artificial181.3 prion181.4 domains181.5 containing181.6 short181.7 (181.8Q22181.9)181.10 and181.11 pathogenically181.12 expanded181.13 (181.14Q62181.15)181.16 polyglutamine181.17 tracts181.18,181.19 either181.20 alone181.21 or181.22 adjacent181.23 to181.24 the181.25 Sup35p181.26 oligopeptide181.27 repeat181.28 (181.29Figure181.30 6A181.31)126.96.36.199
These domains were fused to –M-GFP and –M-C to create polyglutamine inducers and maintainers, respectively.
These182.1 domains182.2 were182.3 fused182.4 to182.5 –182.6M-GFP182.7 and182.8 –182.9M-C182.10 to182.11 create182.12 polyglutamine182.13 inducers182.14 and182.15 maintainers182.16,182.17 respectively188.8.131.52
Q22 inducers did not aggregate upon overexpression, but Q62 inducers (with and without oligopeptide repeats) formed visible foci in [psi–] [PIN+] cells (Figure 6B).
Q22183.1 inducers183.2 did183.3 not183.4 aggregate183.5 upon183.6 overexpression183.7,183.8 but183.9 Q62183.10 inducers183.11 (183.12with183.13 and183.14 without183.15 oligopeptide183.16 repeats183.17)183.18 formed183.19 visible183.20 foci183.21 in183.22 [183.23psi183.24–183.25]183.26 [183.27PIN183.28+183.29]183.30 cells183.31 (183.32Figure183.33 6B183.34)184.108.40.206
Transient overexpression of Q62 inducers had no effect on the Q22 maintainer with the oligopeptide repeat or on the Q62 maintainer lacking the oligopeptide repeat.
Transient184.1 overexpression184.2 of184.3 Q62184.4 inducers184.5 had184.6 no184.7 effect184.8 on184.9 the184.10 Q22184.11 maintainer184.12 with184.13 the184.14 oligopeptide184.15 repeat184.16 or184.17 on184.18 the184.19 Q62184.20 maintainer184.21 lacking184.22 the184.23 oligopeptide184.24 repeat220.127.116.11
However, the Q62 maintainer with an oligopeptide repeat supported prion inheritance, converting to a stable Ade+ state following overexpression of the cognate inducer (Figure 6C).
However185.1,185.2 the185.3 Q62185.4 maintainer185.5 with185.6 an185.7 oligopeptide185.8 repeat185.9 supported185.10 prion185.11 inheritance185.12,185.13 converting185.14 to185.15 a185.16 stable185.17 Ade185.18+185.19 state185.20 following185.21 overexpression185.22 of185.23 the185.24 cognate185.25 inducer185.26 (185.27Figure185.28 6C185.29)18.104.22.168
Several tests confirmed the prion nature of this state, termed [Q+].
Several186.1 tests186.2 confirmed186.3 the186.4 prion186.5 nature186.6 of186.7 this186.8 state186.9,186.10 termed186.11 [186.12Q186.13+186.14]22.214.171.124
Like [PSI+], [Q+] did not require the presence of the inducer plasmid after transient overexpression, was sensitive to GuHCl treatment (Figure 6D), and displayed a dominant, non-Mendelian pattern of inheritance (Figure 6E).
Like187.1 [187.2PSI187.3+187.4]187.5,187.6 [187.7Q187.8+187.9]187.10 did187.11 not187.12 require187.13 the187.14 presence187.15 of187.16 the187.17 inducer187.18 plasmid187.19 after187.20 transient187.21 overexpression187.22,187.23 was187.24 sensitive187.25 to187.26 GuHCl187.27 treatment187.28 (187.29Figure187.30 6D187.31)187.32,187.33 and187.34 displayed187.35 a187.36 dominant187.37,187.38 non-Mendelian187.39 pattern187.40 of187.41 inheritance187.42 (187.43Figure187.44 6E187.45)126.96.36.199
We further tested the specificity of the [Q+] state by introducing a plasmid encoding a noncognate second maintainer into a [Q+] strain (Figure 6F).
We188.1 further188.2 tested188.3 the188.4 specificity188.5 of188.6 the188.7 [188.8Q188.9+188.10]188.11 state188.12 by188.13 introducing188.14 a188.15 plasmid188.16 encoding188.17 a188.18 noncognate188.19 second188.20 maintainer188.21 into188.22 a188.23 [188.24Q188.25+188.26]188.27 strain188.28 (188.29Figure188.30 6F188.31)188.8.131.52
The Q62 maintainer failed to be incorporated into [PSI+] aggregates, causing antisuppression (red); conversely, Sup35p did not enter [Q+] aggregates.
The189.1 Q62189.2 maintainer189.3 failed189.4 to189.5 be189.6 incorporated189.7 into189.8 [189.9PSI189.10+189.11]189.12 aggregates189.13,189.14 causing189.15 antisuppression189.16 (189.17red189.18)189.19;189.20 conversely189.21,189.22 Sup35p189.23 did189.24 not189.25 enter189.26 [189.27Q189.28+189.29]189.30 aggregates184.108.40.206
A number of epigenetic traits in fungi result from the stable inheritance of self-propagating, infectious protein aggregrates (prions) (Uptain and Lindquist 2002).
A191.1 number191.2 of191.3 epigenetic191.4 traits191.5 in191.6 fungi191.7 result191.8 from191.9 the191.10 stable191.11 inheritance191.12 of191.13 self-propagating191.14,191.15 infectious191.16 protein191.17 aggregrates191.18 (191.19prions191.20)191.21 (191.22Uptain191.23 and191.24 Lindquist191.25 2002191.26)220.127.116.11
Prion inheritance requires three sequential events that must keep pace with cell division to preserve the number of heritable prion units, or propagons, per cell (Osherovich and Weissman 2004).
Prion192.1 inheritance192.2 requires192.3 three192.4 sequential192.5 events192.6 that192.7 must192.8 keep192.9 pace192.10 with192.11 cell192.12 division192.13 to192.14 preserve192.15 the192.16 number192.17 of192.18 heritable192.19 prion192.20 units192.21,192.22 or192.23 propagons192.24,192.25 per192.26 cell192.27 (192.28Osherovich192.29 and192.30 Weissman192.31 2004192.32)18.104.22.168
First, prion aggregates must grow in size by incorporating newly synthesized protein.
First193.1,193.2 prion193.3 aggregates193.4 must193.5 grow193.6 in193.7 size193.8 by193.9 incorporating193.10 newly193.11 synthesized193.12 protein22.214.171.124
Next, these enlarged aggregates must be divided into smaller ones through the action of cellular chaperones (Kushnirov and Ter-Avanesyan 1998; Borchsenius et al. 2001; Ness et al. 2002; Kryndushkin et al. 2003).
Next194.1,194.2 these194.3 enlarged194.4 aggregates194.5 must194.6 be194.7 divided194.8 into194.9 smaller194.10 ones194.11 through194.12 the194.13 action194.14 of194.15 cellular194.16 chaperones194.17 (194.18Kushnirov194.19 and194.20 Ter-Avanesyan194.21 1998194.22;194.23 Borchsenius194.24 et194.25 al126.96.36.199 2001194.28;194.29 Ness194.30 et194.31 al188.8.131.52 2002194.34;194.35 Kryndushkin194.36 et194.37 al184.108.40.206 2003194.40)220.127.116.11
Finally, these regenerated propagons must be distributed to mother and daughter cells (Cox et al. 2003); for small, cytoplasmic aggregates, this distribution may occur passively by diffusion during cytokinesis.
Finally195.1,195.2 these195.3 regenerated195.4 propagons195.5 must195.6 be195.7 distributed195.8 to195.9 mother195.10 and195.11 daughter195.12 cells195.13 (195.14Cox195.15 et195.16 al18.104.22.168 2003195.19)195.20;195.21 for195.22 small195.23,195.24 cytoplasmic195.25 aggregates195.26,195.27 this195.28 distribution195.29 may195.30 occur195.31 passively195.32 by195.33 diffusion195.34 during195.35 cytokinesis22.214.171.124
In the present study, we have dissected the prion-forming domains of Sup35p and New1p to discover the sequence elements involved in these steps.
In196.1 the196.2 present196.3 study196.4,196.5 we196.6 have196.7 dissected196.8 the196.9 prion-forming196.10 domains196.11 of196.12 Sup35p196.13 and196.14 New1p196.15 to196.16 discover196.17 the196.18 sequence196.19 elements196.20 involved196.21 in196.22 these196.23 steps126.96.36.199
We have found that these domains consist largely of modular, interchangeable elements that serve distinct functions of prion growth and division or transmission.
We197.1 have197.2 found197.3 that197.4 these197.5 domains197.6 consist197.7 largely197.8 of197.9 modular197.10,197.11 interchangeable197.12 elements197.13 that197.14 serve197.15 distinct197.16 functions197.17 of197.18 prion197.19 growth197.20 and197.21 division197.22 or197.23 transmission188.8.131.52
Aggregation underlies the growth phase of the prion replication cycle (Figure 7A) and occurs through the templated addition of conformationally compatible monomers onto preexisting seeds.
Aggregation198.1 underlies198.2 the198.3 growth198.4 phase198.5 of198.6 the198.7 prion198.8 replication198.9 cycle198.10 (198.11Figure198.12 7A198.13)198.14 and198.15 occurs198.16 through198.17 the198.18 templated198.19 addition198.20 of198.21 conformationally198.22 compatible198.23 monomers198.24 onto198.25 preexisting198.26 seeds184.108.40.206
Like other amyloids, yeast prions display a high specificity for homotypic aggregation (Santoso et al. 2000; Chernoff et al. 2000; Kushnirov et al. 2000; Zadorskii et al. 2000; Nakayashiki et al. 2001).
Like199.1 other199.2 amyloids199.3,199.4 yeast199.5 prions199.6 display199.7 a199.8 high199.9 specificity199.10 for199.11 homotypic199.12 aggregation199.13 (199.14Santoso199.15 et199.16 al220.127.116.11 2000199.19;199.20 Chernoff199.21 et199.22 al18.104.22.168 2000199.25;199.26 Kushnirov199.27 et199.28 al22.214.171.124 2000199.31;199.32 Zadorskii199.33 et199.34 al126.96.36.199 2000199.37;199.38 Nakayashiki199.39 et199.40 al188.8.131.52 2001199.43)184.108.40.206
This discrimination arises from differences in the amino acid sequence and the conformation (Chien and Weissman 2001) of the aggregation-promoting Q/N-rich elements found in each yeast prion protein.
This200.1 discrimination200.2 arises200.3 from200.4 differences200.5 in200.6 the200.7 amino200.8 acid200.9 sequence200.10 and200.11 the200.12 conformation200.13 (200.14Chien200.15 and200.16 Weissman200.17 2001200.18)200.19 of200.20 the200.21 aggregation-promoting200.22 Q/N-rich200.23 elements200.24 found200.25 in200.26 each200.27 yeast200.28 prion200.29 protein220.127.116.11
Aggregation and specificity are dictated by the NYN repeat (residues 70–100) of New1p and by the Q/N-rich amino terminal region (residues 1–57) of Sup35p.
Aggregation201.1 and201.2 specificity201.3 are201.4 dictated201.5 by201.6 the201.7 NYN201.8 repeat201.9 (201.10residues201.11 70201.12–201.13100201.14)201.15 of201.16 New1p201.17 and201.18 by201.19 the201.20 Q/N-rich201.21 amino201.22 terminal201.23 region201.24 (201.25residues201.26 1201.27–201.2857201.29)201.30 of201.31 Sup35p18.104.22.168
[Q+], a Prion Form of Pathogenically Expanded Polyglutamine
[203.1Q203.2+203.3]203.4,203.5 a203.6 Prion203.7 Form203.8 of203.9 Pathogenically203.10 Expanded203.11 Polyglutamine203.12
(A) Schematic illustrating the construction of polyglutamine-derived prion domains. (Op) indicates the presence of the Sup35p oligopeptide repeats (residues 40–124).
(204.1A204.2)204.3 Schematic204.4 illustrating204.5 the204.6 construction204.7 of204.8 polyglutamine-derived204.9 prion204.10 domains22.214.171.124 (204.13Op204.14)204.15 indicates204.16 the204.17 presence204.18 of204.19 the204.20 Sup35p204.21 oligopeptide204.22 repeats204.23 (204.24residues204.25 40204.26–204.27124204.28)126.96.36.199
(B) Fluorescence micrographs of [psi–] [PIN+] strains expressing indicated polyglutamine inducers.
(205.1B205.2)205.3 Fluorescence205.4 micrographs205.5 of205.6 [205.7psi205.8–205.9]205.10 [205.11PIN205.12+205.13]205.14 strains205.15 expressing205.16 indicated205.17 polyglutamine205.18 inducers188.8.131.52
(C) Polyglutamine-based prion inheritance.
(206.1C206.2)206.3 Polyglutamine-based206.4 prion206.5 inheritance184.108.40.206
Strains with indicated inducers and maintainers were plated onto SD-ade and YEPD media to determine the fraction of Ade+ after a standard induction experiment.
Strains207.1 with207.2 indicated207.3 inducers207.4 and207.5 maintainers207.6 were207.7 plated207.8 onto207.9 SD-ade207.10 and207.11 YEPD207.12 media207.13 to207.14 determine207.15 the207.16 fraction207.17 of207.18 Ade207.19+207.20 after207.21 a207.22 standard207.23 induction207.24 experiment220.127.116.11
Interestingly, Q62 inducer forms aggregates but does not promote Ade+ in the Q62(Op) maintainer strain.
Interestingly208.1,208.2 Q62208.3 inducer208.4 forms208.5 aggregates208.6 but208.7 does208.8 not208.9 promote208.10 Ade208.11+208.12 in208.13 the208.14 Q62208.15(208.16Op208.17)208.18 maintainer208.19 strain18.104.22.168
Note that Q62(Op) shows a high rate of spontaneous appearance of Ade+.
Note209.1 that209.2 Q62209.3(209.4Op209.5)209.6 shows209.7 a209.8 high209.9 rate209.10 of209.11 spontaneous209.12 appearance209.13 of209.14 Ade209.15+22.214.171.124
(D) GuHCl sensitivity of the [Q+] state.
(210.1D210.2)210.3 GuHCl210.4 sensitivity210.5 of210.6 the210.7 [210.8Q210.9+210.10]210.11 state126.96.36.199
An Ade+ convertant obtained in (C) was restreaked to lose the inducer plasmid, then grown on GuHCl.
An211.1 Ade211.2+211.3 convertant211.4 obtained211.5 in211.6 (211.7C211.8)211.9 was211.10 restreaked211.11 to211.12 lose211.13 the211.14 inducer211.15 plasmid211.16,211.17 then211.18 grown211.19 on211.20 GuHCl188.8.131.52
Shown are plates before and after GuHCl treatment, along with [psi–] and [PSI+] controls.
Shown212.1 are212.2 plates212.3 before212.4 and212.5 after212.6 GuHCl212.7 treatment212.8,212.9 along212.10 with212.11 [212.12psi212.13–212.14]212.15 and212.16 [212.17PSI212.18+212.19]212.20 controls184.108.40.206
(E) Dominance and non-Mendelian inheritance of [Q+].
(213.1E213.2)213.3 Dominance213.4 and213.5 non-Mendelian213.6 inheritance213.7 of213.8 [213.9Q213.10+213.11]220.127.116.11
See Figure 5C.
See214.1 Figure214.2 5C18.104.22.168
(F) [Q+] does not interact with Sup35p and vice versa.
(215.1F215.2)215.3 [215.4Q215.5+215.6]215.7 does215.8 not215.9 interact215.10 with215.11 Sup35p215.12 and215.13 vice215.14 versa22.214.171.124
[Q+] and [PSI+] strains were transformed with indicated maintainers; mismatches between the maintainer and the chromosomally encoded allele result in antisuppression (red).
[216.1Q216.2+216.3]216.4 and216.5 [216.6PSI216.7+216.8]216.9 strains216.10 were216.11 transformed216.12 with216.13 indicated216.14 maintainers216.15;216.16 mismatches216.17 between216.18 the216.19 maintainer216.20 and216.21 the216.22 chromosomally216.23 encoded216.24 allele216.25 result216.26 in216.27 antisuppression216.28 (216.29red216.30)126.96.36.199
In contrast, the conserved oligopeptide repeat sequence mediates the division and/or segregation phase of prion replication (Figure 7B).
In217.1 contrast217.2,217.3 the217.4 conserved217.5 oligopeptide217.6 repeat217.7 sequence217.8 mediates217.9 the217.10 division217.11 and/or217.12 segregation217.13 phase217.14 of217.15 prion217.16 replication217.17 (217.18Figure217.19 7B217.20)188.8.131.52
In New1p, the NYN repeat alone can aggregate and induce [NU+] but requires an adjacent oligopeptide repeat to form a minimal heritable New1p prion, [NU+]mini.
In218.1 New1p218.2,218.3 the218.4 NYN218.5 repeat218.6 alone218.7 can218.8 aggregate218.9 and218.10 induce218.11 [218.12NU218.13+218.14]218.15 but218.16 requires218.17 an218.18 adjacent218.19 oligopeptide218.20 repeat218.21 to218.22 form218.23 a218.24 minimal218.25 heritable218.26 New1p218.27 prion218.28,218.29 [218.30NU218.31+218.32]218.33mini184.108.40.206
Similarly, in Sup35p, the Q/N-rich amino terminal region mediates aggregation whereas most of the oligopeptide repeats are needed for the inheritance of [PSI+] propagons.
Similarly219.1,219.2 in219.3 Sup35p219.4,219.5 the219.6 Q/N-rich219.7 amino219.8 terminal219.9 region219.10 mediates219.11 aggregation219.12 whereas219.13 most219.14 of219.15 the219.16 oligopeptide219.17 repeats219.18 are219.19 needed219.20 for219.21 the219.22 inheritance219.23 of219.24 [219.25PSI219.26+219.27]219.28 propagons220.127.116.11
Oligopeptide repeats 1 and 2 appear to contribute to both growth and inheritance, consistent with earlier reports that expansion and deletion within this region modulate in vitro polymerization of Sup35p and the appearance of [PSI+] in vivo (Liu and Lindquist 1999).
Oligopeptide220.1 repeats220.2 1220.3 and220.4 2220.5 appear220.6 to220.7 contribute220.8 to220.9 both220.10 growth220.11 and220.12 inheritance220.13,220.14 consistent220.15 with220.16 earlier220.17 reports220.18 that220.19 expansion220.20 and220.21 deletion220.22 within220.23 this220.24 region220.25 modulate220.26 in220.27 vitro220.28 polymerization220.29 of220.30 Sup35p220.31 and220.32 the220.33 appearance220.34 of220.35 [220.36PSI220.37+220.38]220.39 in220.40 vivo220.41 (220.42Liu220.43 and220.44 Lindquist220.45 1999220.46)18.104.22.168
However, the two functions can be distinguished by a point mutant in oligopeptide repeat 2 (PNM2-1), which displays a specific defect in [PSI+] inheritance despite normal aggregation.
However221.1,221.2 the221.3 two221.4 functions221.5 can221.6 be221.7 distinguished221.8 by221.9 a221.10 point221.11 mutant221.12 in221.13 oligopeptide221.14 repeat221.15 2221.16 (221.17PNM2-1221.18)221.19,221.20 which221.21 displays221.22 a221.23 specific221.24 defect221.25 in221.26 [221.27PSI221.28+221.29]221.30 inheritance221.31 despite221.32 normal221.33 aggregation22.214.171.124
Certain [PSI+] variants are resistant to the dominant negative effect of PNM2-1 (Derkatch et al. 1999; King 2001); this suggests that although oligopeptide repeat 2 is critical for the replication of the [PSI+] variant used in our studies, it may be less important for the replication of other Sup35p prion conformations.
Certain222.1 [222.2PSI222.3+222.4]222.5 variants222.6 are222.7 resistant222.8 to222.9 the222.10 dominant222.11 negative222.12 effect222.13 of222.14 PNM2-1222.15 (222.16Derkatch222.17 et222.18 al126.96.36.199 1999222.21;222.22 King222.23 2001222.24)222.25;222.26 this222.27 suggests222.28 that222.29 although222.30 oligopeptide222.31 repeat222.32 2222.33 is222.34 critical222.35 for222.36 the222.37 replication222.38 of222.39 the222.40 [222.41PSI222.42+222.43]222.44 variant222.45 used222.46 in222.47 our222.48 studies222.49,222.50 it222.51 may222.52 be222.53 less222.54 important222.55 for222.56 the222.57 replication222.58 of222.59 other222.60 Sup35p222.61 prion222.62 conformations188.8.131.52
Many studies have established that prion inheritance requires the action of cellular chaperones such as Hsp104p and Hsp70 proteins (reviewed in Osherovich and Weissman 2002), although how these proteins contribute is poorly understood.
Many223.1 studies223.2 have223.3 established223.4 that223.5 prion223.6 inheritance223.7 requires223.8 the223.9 action223.10 of223.11 cellular223.12 chaperones223.13 such223.14 as223.15 Hsp104p223.16 and223.17 Hsp70223.18 proteins223.19 (223.20reviewed223.21 in223.22 Osherovich223.23 and223.24 Weissman223.25 2002223.26)223.27,223.28 although223.29 how223.30 these223.31 proteins223.32 contribute223.33 is223.34 poorly223.35 understood184.108.40.206
We propose that oligopeptide repeats turn nonheritable aggregates into prions by facilitating chaperone-mediated division.
We224.1 propose224.2 that224.3 oligopeptide224.4 repeats224.5 turn224.6 nonheritable224.7 aggregates224.8 into224.9 prions224.10 by224.11 facilitating224.12 chaperone-mediated224.13 division220.127.116.11
Oligopeptide repeats may allow the division of aggregates by providing direct binding sites for chaperones or by altering the conformation of the amyloid core to allow chaperone access.
Oligopeptide225.1 repeats225.2 may225.3 allow225.4 the225.5 division225.6 of225.7 aggregates225.8 by225.9 providing225.10 direct225.11 binding225.12 sites225.13 for225.14 chaperones225.15 or225.16 by225.17 altering225.18 the225.19 conformation225.20 of225.21 the225.22 amyloid225.23 core225.24 to225.25 allow225.26 chaperone225.27 access18.104.22.168
An earlier study established that deletion of residues 22–69 of Sup35p (which include parts of both the Q/N tract and the oligopeptide repeat) interferes with both [PSI+] induction and chaperone-mediated prion disaggregation (Borchsenius et al. 2001).
An226.1 earlier226.2 study226.3 established226.4 that226.5 deletion226.6 of226.7 residues226.8 22226.9–226.1069226.11 of226.12 Sup35p226.13 (226.14which226.15 include226.16 parts226.17 of226.18 both226.19 the226.20 Q/N226.21 tract226.22 and226.23 the226.24 oligopeptide226.25 repeat226.26)226.27 interferes226.28 with226.29 both226.30 [226.31PSI226.32+226.33]226.34 induction226.35 and226.36 chaperone-mediated226.37 prion226.38 disaggregation226.39 (226.40Borchsenius226.41 et226.42 al22.214.171.124 2001226.45)126.96.36.199
Unlike the Δ22–69 mutant, the prion replication defect in PNM2-1 could not be corrected by increasing Hsp104p levels (data not shown), arguing that the mitotic instability of PNM2-1 [PSI+] is not due solely to inadequate Hsp104p binding.
Unlike227.1 the227.2 Δ22227.3–227.469227.5 mutant227.6,227.7 the227.8 prion227.9 replication227.10 defect227.11 in227.12 PNM2-1227.13 could227.14 not227.15 be227.16 corrected227.17 by227.18 increasing227.19 Hsp104p227.20 levels227.21 (227.22data227.23 not227.24 shown227.25)227.26,227.27 arguing227.28 that227.29 the227.30 mitotic227.31 instability227.32 of227.33 PNM2-1227.34 [227.35PSI227.36+227.37]227.38 is227.39 not227.40 due227.41 solely227.42 to227.43 inadequate227.44 Hsp104p227.45 binding188.8.131.52
Model for Prion Growth and Division
Model229.1 for229.2 Prion229.3 Growth229.4 and229.5 Division229.6
(A) During prion growth, polymers seed the incorporation of monomers through interactions between Q/N-rich aggregation sequences (blue).
(230.1A230.2)230.3 During230.4 prion230.5 growth230.6,230.7 polymers230.8 seed230.9 the230.10 incorporation230.11 of230.12 monomers230.13 through230.14 interactions230.15 between230.16 Q/N-rich230.17 aggregation230.18 sequences230.19 (230.20blue230.21)184.108.40.206
Proteins with noncognate aggregation sequences (red) are excluded.
Proteins231.1 with231.2 noncognate231.3 aggregation231.4 sequences231.5 (231.6red231.7)231.8 are231.9 excluded220.127.116.11
(B) The division phase of prion replication requires the oligopeptide repeats (orange), which may facilitate the action of chaperones such as Hsp104p (scimitar) in breaking the polymer into smaller, heritable units.
(232.1B232.2)232.3 The232.4 division232.5 phase232.6 of232.7 prion232.8 replication232.9 requires232.10 the232.11 oligopeptide232.12 repeats232.13 (232.14orange232.15)232.16,232.17 which232.18 may232.19 facilitate232.20 the232.21 action232.22 of232.23 chaperones232.24 such232.25 as232.26 Hsp104p232.27 (232.28scimitar232.29)232.30 in232.31 breaking232.32 the232.33 polymer232.34 into232.35 smaller232.36,232.37 heritable232.38 units18.104.22.168
Our findings help to explain why, among many Q/N-rich proteins in yeast, only a small subset form heritable prions.
Our233.1 findings233.2 help233.3 to233.4 explain233.5 why233.6,233.7 among233.8 many233.9 Q/N-rich233.10 proteins233.11 in233.12 yeast233.13,233.14 only233.15 a233.16 small233.17 subset233.18 form233.19 heritable233.20 prions22.214.171.124
While many Q/N-rich proteins can aggregate when overexpressed (Sondheimer and Lindquist 2000; Derkatch et al. 2001; Osherovich and Weissman 2001), prion inheritance of such aggregates requires that they be divided and passed on to the next generation.
While234.1 many234.2 Q/N-rich234.3 proteins234.4 can234.5 aggregate234.6 when234.7 overexpressed234.8 (234.9Sondheimer234.10 and234.11 Lindquist234.12 2000234.13;234.14 Derkatch234.15 et234.16 al126.96.36.199 2001234.19;234.20 Osherovich234.21 and234.22 Weissman234.23 2001234.24)234.25,234.26 prion234.27 inheritance234.28 of234.29 such234.30 aggregates234.31 requires234.32 that234.33 they234.34 be234.35 divided234.36 and234.37 passed234.38 on234.39 to234.40 the234.41 next234.42 generation188.8.131.52
Although the inheritance of Sup35p and New1p prions is mediated by oligopeptide repeats, other sequences could also serve this purpose.
Although235.1 the235.2 inheritance235.3 of235.4 Sup35p235.5 and235.6 New1p235.7 prions235.8 is235.9 mediated235.10 by235.11 oligopeptide235.12 repeats235.13,235.14 other235.15 sequences235.16 could235.17 also235.18 serve235.19 this235.20 purpose184.108.40.206
Ure2p lacks an oligopeptide repeat; interestingly, many isolates of [URE3] are mitotically unstable in the absence of selection (Schlumpberger et al. 2001).
Ure2p236.1 lacks236.2 an236.3 oligopeptide236.4 repeat236.5;236.6 interestingly236.7,236.8 many236.9 isolates236.10 of236.11 [236.12URE3236.13]236.14 are236.15 mitotically236.16 unstable236.17 in236.18 the236.19 absence236.20 of236.21 selection236.22 (236.23Schlumpberger236.24 et236.25 al220.127.116.11 2001236.28)18.104.22.168
Rnq1p, which underlies [PIN+], also lacks a strict oligopeptide repeat, but a region (residues 218–405) within its prion domain has an amino acid content reminiscent of the oligopeptide repeat sequence (i.e., numerous Q, N, S, Y, and G residues) (Resende et al. 2003).
Rnq1p237.1,237.2 which237.3 underlies237.4 [237.5PIN237.6+237.7]237.8,237.9 also237.10 lacks237.11 a237.12 strict237.13 oligopeptide237.14 repeat237.15,237.16 but237.17 a237.18 region237.19 (237.20residues237.21 218237.22–237.23405237.24)237.25 within237.26 its237.27 prion237.28 domain237.29 has237.30 an237.31 amino237.32 acid237.33 content237.34 reminiscent237.35 of237.36 the237.37 oligopeptide237.38 repeat237.39 sequence237.40 (237.41i22.214.171.124e126.96.36.199,237.46 numerous237.47 Q237.48,237.49 N237.50,237.51 S237.52,237.53 Y237.54,237.55 and237.56 G237.57 residues237.58)237.59 (237.60Resende237.61 et237.62 al188.8.131.52 2003237.65)184.108.40.206
Only two other yeast proteins, YDR210W and YBR016W, have clearly recognizable oligopeptide repeats; both proteins also have Q/N-rich regions.
Only238.1 two238.2 other238.3 yeast238.4 proteins238.5,238.6 YDR210W238.7 and238.8 YBR016W238.9,238.10 have238.11 clearly238.12 recognizable238.13 oligopeptide238.14 repeats238.15;238.16 both238.17 proteins238.18 also238.19 have238.20 Q/N-rich238.21 regions220.127.116.11
YBR016W forms aggregates when overexpressed (Sondheimer and Lindquist 2000), but it is not known whether either protein can maintain a heritable aggregated state.
YBR016W239.1 forms239.2 aggregates239.3 when239.4 overexpressed239.5 (239.6Sondheimer239.7 and239.8 Lindquist239.9 2000239.10)239.11,239.12 but239.13 it239.14 is239.15 not239.16 known239.17 whether239.18 either239.19 protein239.20 can239.21 maintain239.22 a239.23 heritable239.24 aggregated239.25 state18.104.22.168
Although the mammalian prion protein PrP contains a sequence resembling the oligopeptide repeat that can functionally replace one of the Sup35p repeats (Parham et al. 2001), it is unclear whether this sequence is important in the replication of the PrPSc state.
Although240.1 the240.2 mammalian240.3 prion240.4 protein240.5 PrP240.6 contains240.7 a240.8 sequence240.9 resembling240.10 the240.11 oligopeptide240.12 repeat240.13 that240.14 can240.15 functionally240.16 replace240.17 one240.18 of240.19 the240.20 Sup35p240.21 repeats240.22 (240.23Parham240.24 et240.25 al240.26.240.27 2001240.28)240.29,240.30 it240.31 is240.32 unclear240.33 whether240.34 this240.35 sequence240.36 is240.37 important240.38 in240.39 the240.40 replication240.41 of240.42 the240.43 PrPSc240.44 state240.45.240.46
The interchangeable nature of prion domain components allowed us to design novel artificial prions.
The241.1 interchangeable241.2 nature241.3 of241.4 prion241.5 domain241.6 components241.7 allowed241.8 us241.9 to241.10 design241.11 novel241.12 artificial241.13 prions241.14.241.15
The F chimera, consisting of the aggregation sequence of New1p and the oligopeptide repeat of Sup35p, demonstrates that the growth and specificity of prions is largely determined by the Q/N-rich tract, not by the oligopeptide repeat.
The242.1 F242.2 chimera242.3,242.4 consisting242.5 of242.6 the242.7 aggregation242.8 sequence242.9 of242.10 New1p242.11 and242.12 the242.13 oligopeptide242.14 repeat242.15 of242.16 Sup35p242.17,242.18 demonstrates242.19 that242.20 the242.21 growth242.22 and242.23 specificity242.24 of242.25 prions242.26 is242.27 largely242.28 determined242.29 by242.30 the242.31 Q/N-rich242.32 tract242.33,242.34 not242.35 by242.36 the242.37 oligopeptide242.38 repeat242.39.242.40
Despite a sequence derived primarily from Sup35p, the F chimera behaved like New1p rather than like Sup35p.
Despite243.1 a243.2 sequence243.3 derived243.4 primarily243.5 from243.6 Sup35p243.7,243.8 the243.9 F243.10 chimera243.11 behaved243.12 like243.13 New1p243.14 rather243.15 than243.16 like243.17 Sup35p243.18.243.19
The [F+] prion cross-interacted with New1p but not Sup35p.
The244.1 [244.2F244.3+244.4]244.5 prion244.6 cross-interacted244.7 with244.8 New1p244.9 but244.10 not244.11 Sup35p244.12.244.13
Like [NU+], [F+] could be induced in the absence of a prion-promoting (PIN) factor.
Like245.1 [245.2NU245.3+245.4]245.5,245.6 [245.7F245.8+245.9]245.10 could245.11 be245.12 induced245.13 in245.14 the245.15 absence245.16 of245.17 a245.18 prion-promoting245.19 (245.20PIN245.21)245.22 factor245.23.245.24
Finally, [F+] could itself act as a PIN factor, as does [NU+] (data not shown).
Finally246.1,246.2 [246.3F246.4+246.5]246.6 could246.7 itself246.8 act246.9 as246.10 a246.11 PIN246.12 factor246.13,246.14 as246.15 does246.16 [246.17NU246.18+246.19]246.20 (246.21data246.22 not246.23 shown246.24)246.25.246.26
Notably, the NYN repeat of New1p functions as an aggregation module apparently without regard to its position within a protein; this sequence induced prions when overexpressed by itself or with oligopeptide repeats at its N-terminal (in New11–153 and New150–100) or C-terminal regions (in the F chimera).
Notably247.1,247.2 the247.3 NYN247.4 repeat247.5 of247.6 New1p247.7 functions247.8 as247.9 an247.10 aggregation247.11 module247.12 apparently247.13 without247.14 regard247.15 to247.16 its247.17 position247.18 within247.19 a247.20 protein247.21;247.22 this247.23 sequence247.24 induced247.25 prions247.26 when247.27 overexpressed247.28 by247.29 itself247.30 or247.31 with247.32 oligopeptide247.33 repeats247.34 at247.35 its247.36 N-terminal247.37 (247.38in247.39 New11247.40–247.41153247.42 and247.43 New150247.44–247.45100247.46)247.47 or247.48 C-terminal247.49 regions247.50 (247.51in247.52 the247.53 F247.54 chimera247.55)247.56.247.57
These results suggest that aggregation sequences are portable and functionally separable from the oligopeptide repeat, perhaps constituting a structurally discrete amyloid core.
These248.1 results248.2 suggest248.3 that248.4 aggregation248.5 sequences248.6 are248.7 portable248.8 and248.9 functionally248.10 separable248.11 from248.12 the248.13 oligopeptide248.14 repeat248.15,248.16 perhaps248.17 constituting248.18 a248.19 structurally248.20 discrete248.21 amyloid248.22 core248.23.248.24
Indeed, a peptide derived from the amino-terminal region of Sup35p forms a self-seeding amyloid in vitro (Balbirnie et al. 2001).
Indeed249.1,249.2 a249.3 peptide249.4 derived249.5 from249.6 the249.7 amino-terminal249.8 region249.9 of249.10 Sup35p249.11 forms249.12 a249.13 self-seeding249.14 amyloid249.15 in249.16 vitro249.17 (249.18Balbirnie249.19 et249.20 al249.21.249.22 2001249.23)249.24.249.25
A simple aggregation-prone sequence, pathogenically expanded glutamine, also supports prion inheritance when adjacent to the oligopeptide repeat, suggesting that prion domains can consist of little more than a generic, aggregating core sequence and an inheritance-promoting element.
A250.1 simple250.2 aggregation-prone250.3 sequence250.4,250.5 pathogenically250.6 expanded250.7 glutamine250.8,250.9 also250.10 supports250.11 prion250.12 inheritance250.13 when250.14 adjacent250.15 to250.16 the250.17 oligopeptide250.18 repeat250.19,250.20 suggesting250.21 that250.22 prion250.23 domains250.24 can250.25 consist250.26 of250.27 little250.28 more250.29 than250.30 a250.31 generic250.32,250.33 aggregating250.34 core250.35 sequence250.36 and250.37 an250.38 inheritance-promoting250.39 element250.40.250.41
In addition to illuminating the principles of yeast prion domain architecture, artificial prions with distinct specificity may be useful as controllable epigenetic regulators of protein activity.
In251.1 addition251.2 to251.3 illuminating251.4 the251.5 principles251.6 of251.7 yeast251.8 prion251.9 domain251.10 architecture251.11,251.12 artificial251.13 prions251.14 with251.15 distinct251.16 specificity251.17 may251.18 be251.19 useful251.20 as251.21 controllable251.22 epigenetic251.23 regulators251.24 of251.25 protein251.26 activity251.27.251.28
Such prion “switches” can be turned on and off by transient overexpression and genetic repression; for example, the Q prion domain could be fused to other proteins in order to conditionally and reversibly inactivate them independently of [PSI+].
Such252.1 prion252.2 “252.3switches252.4”252.5 can252.6 be252.7 turned252.8 on252.9 and252.10 off252.11 by252.12 transient252.13 overexpression252.14 and252.15 genetic252.16 repression252.17;252.18 for252.19 example252.20,252.21 the252.22 Q252.23 prion252.24 domain252.25 could252.26 be252.27 fused252.28 to252.29 other252.30 proteins252.31 in252.32 order252.33 to252.34 conditionally252.35 and252.36 reversibly252.37 inactivate252.38 them252.39 independently252.40 of252.41 [252.42PSI252.43+252.44]252.45.252.46
It may also be possible to design additional artificial yeast prion domains whose aggregation is driven by non-Q/N-rich amyloidogenic proteins such as the AΒ peptide that accumulates in Alzheimer's disease (Koo et al. 1999) or the mammalian prion protein PrP (Cohen and Prusiner 1998).
It253.1 may253.2 also253.3 be253.4 possible253.5 to253.6 design253.7 additional253.8 artificial253.9 yeast253.10 prion253.11 domains253.12 whose253.13 aggregation253.14 is253.15 driven253.16 by253.17 non-Q/N-rich253.18 amyloidogenic253.19 proteins253.20 such253.21 as253.22 the253.23 AΒ peptide253.24 that253.25 accumulates253.26 in253.27 Alzheimer's253.28 disease253.29 (253.30Koo253.31 et253.32 al253.33.253.34 1999253.35)253.36 or253.37 the253.38 mammalian253.39 prion253.40 protein253.41 PrP253.42 (253.43Cohen253.44 and253.45 Prusiner253.46 1998253.47)253.48.253.49
Such artificial prions could serve as models for aggregate–chaperone interactions in metazoans and could provide a genetic system for the high-throughput screening of modulators of human aggregation diseases.
Such254.1 artificial254.2 prions254.3 could254.4 serve254.5 as254.6 models254.7 for254.8 aggregate254.9–254.10chaperone254.11 interactions254.12 in254.13 metazoans254.14 and254.15 could254.16 provide254.17 a254.18 genetic254.19 system254.20 for254.21 the254.22 high-throughput254.23 screening254.24 of254.25 modulators254.26 of254.27 human254.28 aggregation254.29 diseases254.30.254.31
Materials and Methods
Materials255.1 and255.2 Methods255.3
Yeast strains and methods
Yeast256.1 strains256.2 and256.3 methods256.4
Derivatives of W303 (Osherovich and Weissman 2001), with the initial genotypes ade1-14, his3-11,15, leu2-3, trp1-1, and ura3-1, were used throughout unless otherwise noted; all strains were [PIN+].
Derivatives257.1 of257.2 W303257.3 (257.4Osherovich257.5 and257.6 Weissman257.7 2001257.8)257.9,257.10 with257.11 the257.12 initial257.13 genotypes257.14 ade1-14257.15,257.16 his3-11257.17,257.1815257.19,257.20 leu2-3257.21,257.22 trp1-1257.23,257.24 and257.25 ura3-1257.26,257.27 were257.28 used257.29 throughout257.30 unless257.31 otherwise257.32 noted257.33;257.34 all257.35 strains257.36 were257.37 [257.38PIN257.39+257.40]257.41.257.42
Strain numbers, with indicated genotypic differences, are as follows: YJW 584 [psi–] MATα, YJW 508 [PSI+] MATα, YJW 716 [nu–] MATα sup35::TRP1 pRS315SpNew11–153-M-C, YJW 717 [NU+] MATα sup35::TRP1 pRS315SpNew11–153-M-C, YJW 844 [f –] MATα sup35::F-M-C C.g. HIS3, YJW 881 [F+] MATα sup35::F-M-C C.g. HIS3, YJW 867 [q–] MATα sup35::Q-M-C C.g. HIS3, YJW 868 [Q+] MATα sup35::Q-M-C C.g. HIS3.
Strain258.1 numbers258.2,258.3 with258.4 indicated258.5 genotypic258.6 differences258.7,258.8 are258.9 as258.10 follows258.11:258.12 YJW258.13 584258.14 [258.15psi258.16–258.17]258.18 MATα258.19,258.20 YJW258.21 508258.22 [258.23PSI258.24+258.25]258.26 MATα258.27,258.28 YJW258.29 716258.30 [258.31nu258.32–258.33]258.34 MATα258.35 sup35258.36:258.37:258.38TRP1258.39 pRS315SpNew11258.40–258.41153-M-C258.42,258.43 YJW258.44 717258.45 [258.46NU258.47+258.48]258.49 MATα258.50 sup35258.51:258.52:258.53TRP1258.54 pRS315SpNew11258.55–258.56153-M-C258.57,258.58 YJW258.59 844258.60 [258.61f258.62 –258.63]258.64 MATα258.65 sup35258.66:258.67:258.68F-M-C258.69 C258.70.258.71g258.72.258.73 HIS3258.74,258.75 YJW258.76 881258.77 [258.78F258.79+258.80]258.81 MATα258.82 sup35258.83:258.84:258.85F-M-C258.86 C258.87.258.88g258.89.258.90 HIS3258.91,258.92 YJW258.93 867258.94 [258.95q258.96–258.97]258.98 MATα258.99 sup35258.100:258.101:258.102Q-M-C258.103 C258.104.258.105g258.106.258.107 HIS3258.108,258.109 YJW258.110 868258.111 [258.112Q258.113+258.114]258.115 MATα258.116 sup35258.117:258.118:258.119Q-M-C258.120 C258.121.258.122g258.123.258.124 HIS3258.125.258.126
Maintainer plasmids used in Figure 3 (see plasmid and gene replacement construction, below) were introduced by plasmid shuffling into YJW 716 or YJW 753 ([PSI+] MATα sup35::TRP1 pRS316SpSUP35), followed by loss of the maintainer spontaneously or through 5-FOA counterselection.
Maintainer259.1 plasmids259.2 used259.3 in259.4 Figure259.5 3259.6 (259.7see259.8 plasmid259.9 and259.10 gene259.11 replacement259.12 construction259.13,259.14 below259.15)259.16 were259.17 introduced259.18 by259.19 plasmid259.20 shuffling259.21 into259.22 YJW259.23 716259.24 or259.25 YJW259.26 753259.27 (259.28[259.29PSI259.30+259.31]259.32 MATα259.33 sup35259.34:259.35:259.36TRP1259.37 pRS316SpSUP35259.38)259.39,259.40 followed259.41 by259.42 loss259.43 of259.44 the259.45 maintainer259.46 spontaneously259.47 or259.48 through259.49 5-FOA259.50 counterselection259.51.259.52
The PNM2-1 strain in Figure 4 was generated in this manner and was subsequently restreaked on SD-ade to select for [PSI+].
The260.1 PNM2-1260.2 strain260.3 in260.4 Figure260.5 4260.6 was260.7 generated260.8 in260.9 this260.10 manner260.11 and260.12 was260.13 subsequently260.14 restreaked260.15 on260.16 SD-ade260.17 to260.18 select260.19 for260.20 [260.21PSI260.22+260.23]260.24.260.25
HIS3-marked oligopeptide repeat truncations and PNM2-1 maintainers were from Parham et al. (2001); all other Sup35p and New1p maintainers were marked with LEU2.
HIS3-marked261.1 oligopeptide261.2 repeat261.3 truncations261.4 and261.5 PNM2-1261.6 maintainers261.7 were261.8 from261.9 Parham261.10 et261.11 al261.12.261.13 (261.142001261.15)261.16;261.17 all261.18 other261.19 Sup35p261.20 and261.21 New1p261.22 maintainers261.23 were261.24 marked261.25 with261.26 LEU2261.27.261.28
The [f–] strain was generated by “gamma” chromosomal integration of pRS306 F-M-C into the SUP35 locus of YJW 584; excision of the wild-type gene was confirmed by PCR of Ade- colonies arising from subsequent growth on 5-FOA.
The262.1 [262.2f262.3–262.4]262.5 strain262.6 was262.7 generated262.8 by262.9 “262.10gamma262.11”262.12 chromosomal262.13 integration262.14 of262.15 pRS306262.16 F-M-C262.17 into262.18 the262.19 SUP35262.20 locus262.21 of262.22 YJW262.23 584262.24;262.25 excision262.26 of262.27 the262.28 wild-type262.29 gene262.30 was262.31 confirmed262.32 by262.33 PCR262.34 of262.35 Ade- colonies262.36 arising262.37 from262.38 subsequent262.39 growth262.40 on262.41 5-FOA262.42.262.43
The [q–] strain was made by “omega” chromosomal gene replacement (Kitada et al. 1995) of SUP35 with a C.glabrata HIS3-marked –M-C variant (with or without oligopeptide repeats) into the SUP35 locus of a diploid [PSI+] [PIN+] strain.
The263.1 [263.2q263.3–263.4]263.5 strain263.6 was263.7 made263.8 by263.9 “263.10omega263.11”263.12 chromosomal263.13 gene263.14 replacement263.15 (263.16Kitada263.17 et263.18 al263.19.263.20 1995263.21)263.22 of263.23 SUP35263.24 with263.25 a263.26 C263.27.263.28glabrata263.29 HIS3-marked263.30 –263.31M-C263.32 variant263.33 (263.34with263.35 or263.36 without263.37 oligopeptide263.38 repeats263.39)263.40 into263.41 the263.42 SUP35263.43 locus263.44 of263.45 a263.46 diploid263.47 [263.48PSI263.49+263.50]263.51 [263.52PIN263.53+263.54]263.55 strain263.56.263.57
After sporulation, gene replacement was confirmed by PCR and by loss of [PSI+] in half of the haploid progeny.
After264.1 sporulation264.2,264.3 gene264.4 replacement264.5 was264.6 confirmed264.7 by264.8 PCR264.9 and264.10 by264.11 loss264.12 of264.13 [264.14PSI264.15+264.16]264.17 in264.18 half264.19 of264.20 the264.21 haploid264.22 progeny264.23.264.24
Yeast culture methods were according to standard procedures (Sherman 1991), but YEPD-medium plates contained 1/4 of the standard amount of yeast extract to accentuate color phenotypes.
Yeast265.1 culture265.2 methods265.3 were265.4 according265.5 to265.6 standard265.7 procedures265.8 (265.9Sherman265.10 1991265.11)265.12,265.13 but265.14 YEPD-medium265.15 plates265.16 contained265.17 1/4265.18 of265.19 the265.20 standard265.21 amount265.22 of265.23 yeast265.24 extract265.25 to265.26 accentuate265.27 color265.28 phenotypes265.29.265.30
For prion curing, strains were grown on YEPD medium plus 3 mM GuHCl, then restreaked onto YEPD medium.
For266.1 prion266.2 curing266.3,266.4 strains266.5 were266.6 grown266.7 on266.8 YEPD266.9 medium266.10 plus266.11 3266.12 mM266.13 GuHCl266.14,266.15 then266.16 restreaked266.17 onto266.18 YEPD266.19 medium266.20.266.21
Plasmid and gene replacement construction
Plasmid267.1 and267.2 gene267.3 replacement267.4 construction267.5
The modular SUP35 cloning system described in previous reports was used throughout (Santoso et al. 2000; Osherovich and Weissman 2001).
The268.1 modular268.2 SUP35268.3 cloning268.4 system268.5 described268.6 in268.7 previous268.8 reports268.9 was268.10 used268.11 throughout268.12 (268.13Santoso268.14 et268.15 al268.16.268.17 2000268.18;268.19 Osherovich268.20 and268.21 Weissman268.22 2001268.23)268.24.268.25
All plasmids are derived from Sikorski and Hieter (1989); sequence files of all constructs are available as a web supplement (Data S1).
All269.1 plasmids269.2 are269.3 derived269.4 from269.5 Sikorski269.6 and269.7 Hieter269.8 (269.91989269.10)269.11;269.12 sequence269.13 files269.14 of269.15 all269.16 constructs269.17 are269.18 available269.19 as269.20 a269.21 web269.22 supplement269.23 (269.24Data269.25 S1269.26)269.27.269.28
Maintainer plasmids are low-copy CEN/ARS (pRS31x series) with the native SUP35 promoter (Sp) driving the expression of the indicated prion domain followed by the M and C domains of Sup35p.
Maintainer270.1 plasmids270.2 are270.3 low-copy270.4 CEN/ARS270.5 (270.6pRS31x270.7 series270.8)270.9 with270.10 the270.11 native270.12 SUP35270.13 promoter270.14 (270.15Sp270.16)270.17 driving270.18 the270.19 expression270.20 of270.21 the270.22 indicated270.23 prion270.24 domain270.25 followed270.26 by270.27 the270.28 M270.29 and270.30 C270.31 domains270.32 of270.33 Sup35p270.34.270.35
Inducer plasmids are high-copy 2µ (pRS42x series) with the inducible CUP1 promoter (Cp) driving the expression of the indicated prion domain followed by the Sup35p M domain and GFP.
Inducer271.1 plasmids271.2 are271.3 high-copy271.4 2271.5µ271.6 (271.7pRS42x271.8 series271.9)271.10 with271.11 the271.12 inducible271.13 CUP1271.14 promoter271.15 (271.16Cp271.17)271.18 driving271.19 the271.20 expression271.21 of271.22 the271.23 indicated271.24 prion271.25 domain271.26 followed271.27 by271.28 the271.29 Sup35p271.30 M271.31 domain271.32 and271.33 GFP271.34.271.35
New1p inducers did not include the Sup35p M domain.
New1p272.1 inducers272.2 did272.3 not272.4 include272.5 the272.6 Sup35p272.7 M272.8 domain272.9.272.10
For polyglutamine constructs, polyglutamine tracts (22 and 62) were amplified out of the MJDtr constructs used in an earlier study (Osherovich and Weissman 2001).
For273.1 polyglutamine273.2 constructs273.3,273.4 polyglutamine273.5 tracts273.6 (273.722273.8 and273.9 62273.10)273.11 were273.12 amplified273.13 out273.14 of273.15 the273.16 MJDtr273.17 constructs273.18 used273.19 in273.20 an273.21 earlier273.22 study273.23 (273.24Osherovich273.25 and273.26 Weissman273.27 2001273.28)273.29.273.30
To permit amplification, primers contained sequences homologous to several codons adjacent to the 5' and 3' ends of the polyglutamine tracts plus an initiator ATG codon.
To274.1 permit274.2 amplification274.3,274.4 primers274.5 contained274.6 sequences274.7 homologous274.8 to274.9 several274.10 codons274.11 adjacent274.12 to274.13 the274.14 5' and274.15 3' ends274.16 of274.17 the274.18 polyglutamine274.19 tracts274.20 plus274.21 an274.22 initiator274.23 ATG274.24 codon274.25.274.26
Thus, the polyglutamine sequences read MAYFEK(Q22/62)DLSG.
Thus275.1,275.2 the275.3 polyglutamine275.4 sequences275.5 read275.6 MAYFEK275.7(275.8Q22/62275.9)275.10DLSG275.11.275.12
The resulting PCR fragments were cloned into maintainer and inducer plasmids, which were used as templates for gene replacement PCR (see yeast strains and methods, above).
The276.1 resulting276.2 PCR276.3 fragments276.4 were276.5 cloned276.6 into276.7 maintainer276.8 and276.9 inducer276.10 plasmids276.11,276.12 which276.13 were276.14 used276.15 as276.16 templates276.17 for276.18 gene276.19 replacement276.20 PCR276.21 (276.22see276.23 yeast276.24 strains276.25 and276.26 methods276.27,276.28 above276.29)276.30.276.31
In vivo prion assays
In277.1 vivo277.2 prion277.3 assays277.4
For aggregation, inducers were overexpressed by growth of cells in selective medium with 50 µM CuSO4 until the culture reached stationary phase; cells were then examined by fluorescent microscopy (Zeiss Axiovert, Zeiss, Oberkochen, Germany; Metamorph imaging software, Universal Imaging Corporation, Downingtown, Pennsylvania, United States).
For278.1 aggregation278.2,278.3 inducers278.4 were278.5 overexpressed278.6 by278.7 growth278.8 of278.9 cells278.10 in278.11 selective278.12 medium278.13 with278.14 50278.15 µM278.16 CuSO4278.17 until278.18 the278.19 culture278.20 reached278.21 stationary278.22 phase278.23;278.24 cells278.25 were278.26 then278.27 examined278.28 by278.29 fluorescent278.30 microscopy278.31 (278.32Zeiss278.33 Axiovert278.34,278.35 Zeiss278.36,278.37 Oberkochen278.38,278.39 Germany278.40;278.41 Metamorph278.42 imaging278.43 software278.44,278.45 Universal278.46 Imaging278.47 Corporation278.48,278.49 Downingtown278.50,278.51 Pennsylvania278.52,278.53 United278.54 States278.55)278.56.278.57
Unless otherwise noted, cultures displaying 10% or more cells with foci were scored as positive.
Unless279.1 otherwise279.2 noted279.3,279.4 cultures279.5 displaying279.6 10% or279.7 more279.8 cells279.9 with279.10 foci279.11 were279.12 scored279.13 as279.14 positive279.15.279.16
For induction, dilutions of the above cultures were plated onto SD-ade and YEPD media to determine percentage of Ade+.
For280.1 induction280.2,280.3 dilutions280.4 of280.5 the280.6 above280.7 cultures280.8 were280.9 plated280.10 onto280.11 SD-ade280.12 and280.13 YEPD280.14 media280.15 to280.16 determine280.17 percentage280.18 of280.19 Ade280.20+280.21.280.22
In qualitative assessments, strains were scored as positive if 5% or more of plated cells grew on SD-ade medium after 5 d.
In281.1 qualitative281.2 assessments281.3,281.4 strains281.5 were281.6 scored281.7 as281.8 positive281.9 if281.10 5% or281.11 more281.12 of281.13 plated281.14 cells281.15 grew281.16 on281.17 SD-ade281.18 medium281.19 after281.20 5281.21 d281.22.281.23
In [NU+] maintenance experiments, strains with indicated maintainers were tested for the ability to support an Ade+ state following New11–153-GFP overexpression.
In282.1 [282.2NU282.3+282.4]282.5 maintenance282.6 experiments282.7,282.8 strains282.9 with282.10 indicated282.11 maintainers282.12 were282.13 tested282.14 for282.15 the282.16 ability282.17 to282.18 support282.19 an282.20 Ade282.21+282.22 state282.23 following282.24 New11282.25–282.26153-GFP282.27 overexpression282.28.282.29
In [PSI+] maintenance experiments, strains that began as [PSI+] were tested for Ade+ after plasmid shuffle gene replacement with the indicated maintainer.
In283.1 [283.2PSI283.3+283.4]283.5 maintenance283.6 experiments283.7,283.8 strains283.9 that283.10 began283.11 as283.12 [283.13PSI283.14+283.15]283.16 were283.17 tested283.18 for283.19 Ade283.20+283.21 after283.22 plasmid283.23 shuffle283.24 gene283.25 replacement283.26 with283.27 the283.28 indicated283.29 maintainer283.30.283.31
For decoration, a [PSI+] [PIN+] strain was transformed with the indicated inducers, grown in selective medium with 50 µM CuSO4, and examined by fluorescence microscopy during midlogarithmic phase.
For284.1 decoration284.2,284.3 a284.4 [284.5PSI284.6+284.7]284.8 [284.9PIN284.10+284.11]284.12 strain284.13 was284.14 transformed284.15 with284.16 the284.17 indicated284.18 inducers284.19,284.20 grown284.21 in284.22 selective284.23 medium284.24 with284.25 50284.26 µM284.27 CuSO4284.28,284.29 and284.30 examined284.31 by284.32 fluorescence284.33 microscopy284.34 during284.35 midlogarithmic284.36 phase284.37.284.38
Propagon counts were performed as described in Cox et al. (2003).
Propagon285.1 counts285.2 were285.3 performed285.4 as285.5 described285.6 in285.7 Cox285.8 et285.9 al285.10.285.11 (285.122003285.13)285.14.285.15
For the antisuppression assay, indicated strains were transformed with a second, differently marked maintainer plasmid, and color phenotypes were assayed on medium selective for both plasmids.
For286.1 the286.2 antisuppression286.3 assay286.4,286.5 indicated286.6 strains286.7 were286.8 transformed286.9 with286.10 a286.11 second286.12,286.13 differently286.14 marked286.15 maintainer286.16 plasmid286.17,286.18 and286.19 color286.20 phenotypes286.21 were286.22 assayed286.23 on286.24 medium286.25 selective286.26 for286.27 both286.28 plasmids286.29.286.30
In vitro prion assays
In287.1 vitro287.2 prion287.3 assays287.4
Centrifugation was performed as described in Ness et al. (2002).
Centrifugation288.1 was288.2 performed288.3 as288.4 described288.5 in288.6 Ness288.7 et288.8 al288.9.288.10 (288.112002288.12)288.13.288.14
Immunoblots were visualized with MT130 anti-Sup35p N-M domain serum.
Immunoblots289.1 were289.2 visualized289.3 with289.4 MT130289.5 anti-Sup35p289.6 N-M289.7 domain289.8 serum289.9.289.10
For the polymerization of PNM2-1, the PNM2-1 N and M domains were cloned as 7-histidine fusions into pAED4 and expressed and purified as described in DePace et al. (1998).
For290.1 the290.2 polymerization290.3 of290.4 PNM2-1290.5,290.6 the290.7 PNM2-1290.8 N290.9 and290.10 M290.11 domains290.12 were290.13 cloned290.14 as290.15 7-histidine290.16 fusions290.17 into290.18 pAED4290.19 and290.20 expressed290.21 and290.22 purified290.23 as290.24 described290.25 in290.26 DePace290.27 et290.28 al290.29.290.30 (290.311998290.32)290.33.290.34
Thioflavin-T binding was conducted as in Chien et al. (2003).
Thioflavin-T291.1 binding291.2 was291.3 conducted291.4 as291.5 in291.6 Chien291.7 et291.8 al291.9.291.10 (291.112003291.12)291.13.291.14
The slope of early (0–6 min) dye binding was obtained from seeded polymerization reactions conducted in triplicate.
The292.1 slope292.2 of292.3 early292.4 (292.50292.6–292.76292.8 min292.9)292.10 dye292.11 binding292.12 was292.13 obtained292.14 from292.15 seeded292.16 polymerization292.17 reactions292.18 conducted292.19 in292.20 triplicate292.21.292.22
To correct for a difference in dye binding between wild-type and PNM2-1 protein, these values were normalized to the end point (90 min) maximum signal for each protein.
To293.1 correct293.2 for293.3 a293.4 difference293.5 in293.6 dye293.7 binding293.8 between293.9 wild-type293.10 and293.11 PNM2-1293.12 protein293.13,293.14 these293.15 values293.16 were293.17 normalized293.18 to293.19 the293.20 end293.21 point293.22 (293.2390293.24 min293.25)293.26 maximum293.27 signal293.28 for293.29 each293.30 protein293.31.293.32
Monomer concentrations were 2.5µM.
Monomer294.1 concentrations294.2 were294.3 2294.4.294.55294.6µM294.7.294.8
Osherovich LZ, Cox BS, Tuite MF, Weissman JS (2004) Dissection and Design of Yeast Prions. PLoS Biol 2(4): e86 DOI:10.1371/journal.pbio.0020086.