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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 

Introduction

Introduction_1.1

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).

The_2.1 aggregation_2.2 of_2.3 misfolded_2.4 proteins_2.5 underlies_2.6 a_2.7 diverse_2.8 range_2.9 of_2.10 human_2.11 diseases_2.12,_2.13 including_2.14 sporadic_2.15 amyloidoses_2.16 such_2.17 as_2.18 Alzheimer's_2.19 disease_2.20 and_2.21 hereditary_2.22 neuropathies_2.23 such_2.24 as_2.25 Huntington's_2.26 disease_2.27 (_2.28Dobson_2.29 1999_2.30)_2.31._2.32

Prions are a special class of protein aggregates that replicate their conformation and spread infectiously (Prusiner 1998).

Prions_3.1 are_3.2 a_3.3 special_3.4 class_3.5 of_3.6 protein_3.7 aggregates_3.8 that_3.9 replicate_3.10 their_3.11 conformation_3.12 and_3.13 spread_3.14 infectiously_3.15 (_3.16Prusiner_3.17 1998_3.18)_3.19._3.20

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).

After_4.1 the_4.2 discovery_4.3 that_4.4 prion_4.5 aggregates_4.6 are_4.7 responsible_4.8 for_4.9 the_4.10 mammalian_4.11 transmissible_4.12 spongiform_4.13 encephalopathies_4.14,_4.15 several_4.16 epigenetically_4.17 heritable_4.18 traits_4.19 in_4.20 fungi_4.21 were_4.22 also_4.23 found_4.24 to_4.25 depend_4.26 on_4.27 a_4.28 prion_4.29 mechanism_4.30 (_4.31Wickner_4.32 1994_4.33;_4.34 Uptain_4.35 and_4.36 Lindquist_4.37 2002_4.38;_4.39 Osherovich_4.40 and_4.41 Weissman_4.42 2004_4.43)_4.44._4.45

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).

In_5.1 Saccharomyces_5.2 cerevisiae_5.3 and_5.4 Podospora_5.5 anserina_5.6,_5.7 prions_5.8 are_5.9 transmitted_5.10 from_5.11 cell_5.12 to_5.13 cell_5.14 through_5.15 mating_5.16 and_5.17 cell_5.18 division_5.19,_5.20 resulting_5.21 in_5.22 readily_5.23 assayed_5.24 phenotypes_5.25 with_5.26 a_5.27 non-Mendelian_5.28 pattern_5.29 of_5.30 inheritance_5.31 (_5.32Liebman_5.33 and_5.34 Derkatch_5.35 1999_5.36)_5.37._5.38

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.

The_6.1 yeast_6.2 non-Mendelian_6.3 factors_6.4 [_6.5PSI_6.6+_6.7]_6.8 (_6.9Cox_6.10 1965_6.11)_6.12 and_6.13 [_6.14URE3_6.15]_6.16 (_6.17Lacroute_6.18 1971_6.19)_6.20,_6.21 which_6.22 are_6.23 prion_6.24 forms_6.25 of_6.26 the_6.27 translation_6.28 termination_6.29 factor_6.30 Sup35p_6.31 and_6.32 the_6.33 transcriptional_6.34 activator_6.35 Ure2p_6.36,_6.37 respectively_6.38,_6.39 have_6.40 served_6.41 as_6.42 useful_6.43 models_6.44 for_6.45 the_6.46 formation_6.47 and_6.48 replication_6.49 of_6.50 heritable_6.51 protein_6.52 aggregates_6.53._6.54

Prion forms of Sup35p and Ure2p lead to defects in their respective biochemical activities (translation termination and nitrogen catabolite repression).

Prion_7.1 forms_7.2 of_7.3 Sup35p_7.4 and_7.5 Ure2p_7.6 lead_7.7 to_7.8 defects_7.9 in_7.10 their_7.11 respective_7.12 biochemical_7.13 activities_7.14 (_7.15translation_7.16 termination_7.17 and_7.18 nitrogen_7.19 catabolite_7.20 repression_7.21)_7.22._7.23

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).

Mutational_8.1 analysis_8.2 has_8.3 shown_8.4 the_8.5 glutamine/asparagine-rich_8.6 (_8.7Q/N-rich_8.8)_8.9 amino-terminal_8.10 (_8.11N_8.12)_8.13 domains_8.14 of_8.15 these_8.16 proteins_8.17 to_8.18 be_8.19 critical_8.20 for_8.21 prion_8.22 behavior_8.23 (_8.24Ter-Avanesyan_8.25 et_8.26 al_8.27._8.28 1993_8.29;_8.30 Masison_8.31 and_8.32 Wickner_8.33 1995_8.34;_8.35 Patino_8.36 et_8.37 al_8.38._8.39 1996_8.40;_8.41 Paushkin_8.42 et_8.43 al_8.44._8.45 1996_8.46;_8.47 DePace_8.48 et_8.49 al_8.50._8.51 1998_8.52)_8.53._8.54

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).

In_9.1 vitro_9.2,_9.3 these_9.4 Q/N-rich_9.5 domains_9.6 form_9.7 self-seeding_9.8,_9.9 Β-sheet-rich_9.10 amyloid_9.11 fibrils_9.12 similar_9.13 to_9.14 those_9.15 associated_9.16 with_9.17 Alzheimer's_9.18 and_9.19 Huntington's_9.20 diseases_9.21 (_9.22Glover_9.23 et_9.24 al_9.25._9.26 1997_9.27;_9.28 King_9.29 et_9.30 al_9.31._9.32 1997_9.33;_9.34 Taylor_9.35 et_9.36 al_9.37._9.38 1999_9.39)_9.40._9.41

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).

The_10.1 autocatalytic_10.2 aggregation_10.3 of_10.4 yeast_10.5 prion_10.6 proteins_10.7 often_10.8 shows_10.9 a_10.10 high_10.11 specificity_10.12 for_10.13 like_10.14 molecules_10.15;_10.16 for_10.17 example_10.18,_10.19 Sup35p_10.20 N_10.21 domains_10.22 from_10.23 different_10.24 yeast_10.25 species_10.26 form_10.27 prion_10.28 aggregates_10.29 that_10.30 preferentially_10.31 interact_10.32 with_10.33 molecules_10.34 of_10.35 their_10.36 own_10.37 kind_10.38 (_10.39Santoso_10.40 et_10.41 al_10.42._10.43 2000_10.44;_10.45 Chernoff_10.46 et_10.47 al_10.48._10.49 2000_10.50;_10.51 Kushnirov_10.52 et_10.53 al_10.54._10.55 2000_10.56;_10.57 Zadorskii_10.58 et_10.59 al_10.60._10.61 2000_10.62;_10.63 Nakayashiki_10.64 et_10.65 al_10.66._10.67 2001_10.68)_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).

[_11.1PSI_11.2+_11.3]_11.4 and_11.5 [_11.6URE3_11.7]_11.8 can_11.9 be_11.10 eliminated_11.11 by_11.12 transient_11.13 growth_11.14 in_11.15 the_11.16 presence_11.17 of_11.18 guanidine_11.19 hydrochloride_11.20 (_11.21GuHCl_11.22)_11.23,_11.24 which_11.25 “_11.26cures_11.27”_11.28 cells_11.29 of_11.30 prions_11.31 by_11.32 inhibiting_11.33 Hsp104p_11.34,_11.35 a_11.36 molecular_11.37 chaperone_11.38 needed_11.39 for_11.40 prion_11.41 replication_11.42 (_11.43Chernoff_11.44 et_11.45 al_11.46._11.47 1995_11.48;_11.49 Jung_11.50 et_11.51 al_11.52._11.53 2002_11.54;_11.55 Ness_11.56 et_11.57 al_11.58._11.59 2002_11.60)_11.61._11.62

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).

A_12.1 surprisingly_12.2 large_12.3 number_12.4 of_12.5 proteins_12.6 in_12.7 S_12.8._12.9 cerevisiae_12.10 and_12.11 other_12.12 eukaryotes_12.13 have_12.14 lengthy_12.15 Q/N-rich_12.16 tracts_12.17 ostensibly_12.18 similar_12.19 to_12.20 those_12.21 found_12.22 in_12.23 the_12.24 prion-forming_12.25 domains_12.26 of_12.27 Sup35p_12.28 and_12.29 Ure2p_12.30 (_12.31Michelitsch_12.32 and_12.33 Weissman_12.34 2000_12.35)_12.36._12.37

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).

From_13.1 among_13.2 these_13.3,_13.4 we_13.5 and_13.6 another_13.7 group_13.8 identified_13.9 two_13.10 novel_13.11 proteins_13.12,_13.13 New1p_13.14 and_13.15 Rnq1p_13.16,_13.17 with_13.18 prion-forming_13.19 domains_13.20 resembling_13.21 those_13.22 of_13.23 Sup35p_13.24 and_13.25 Ure2p_13.26 (_13.27Santoso_13.28 et_13.29 al_13.30._13.31 2000_13.32;_13.33 Sondheimer_13.34 and_13.35 Lindquist_13.36 2000_13.37)_13.38._13.39

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.

When_14.1 these_14.2 Q/N-rich_14.3 domains_14.4 were_14.5 fused_14.6 to_14.7 green_14.8 fluorescent_14.9 protein_14.10 (_14.11GFP_14.12)_14.13 and_14.14 overexpressed_14.15,_14.16 they_14.17 formed_14.18 visible_14.19 aggregates_14.20 resembling_14.21 those_14.22 of_14.23 GFP-labeled_14.24 Sup35p_14.25 in_14.26 [_14.27PSI_14.28+_14.29]_14.30 cells_14.31._14.32

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+].

Fusion_15.1 proteins_15.2 in_15.3 which_15.4 these_15.5 domains_15.6 were_15.7 introduced_15.8 in_15.9 place_15.10 of_15.11 the_15.12 Sup35p_15.13 prion_15.14 domain_15.15 could_15.16 support_15.17 distinct_15.18,_15.19 self-specific_15.20 prion_15.21 states_15.22 that_15.23 recapitulated_15.24 the_15.25 translation_15.26 termination_15.27 defect_15.28 associated_15.29 with_15.30 [_15.31PSI_15.32+_15.33]_15.34._15.35

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).

Rnq1p_16.1 was_16.2 later_16.3 shown_16.4 to_16.5 underlie_16.6 a_16.7 naturally_16.8 occurring_16.9 prion_16.10 called_16.11 [_16.12PIN_16.13+_16.14]_16.15,_16.16 which_16.17 promotes_16.18 the_16.19 aggregation_16.20 of_16.21 Q/N-rich_16.22 proteins_16.23 such_16.24 as_16.25 Sup35p_16.26;_16.27 overexpressed_16.28 Sup35p_16.29 forms_16.30 aggregates_16.31 and_16.32 stimulates_16.33 the_16.34 appearance_16.35 of_16.36 [_16.37PSI_16.38+_16.39]_16.40 only_16.41 in_16.42 [_16.43PIN_16.44+_16.45]_16.46 strains_16.47 (_16.48Derkatch_16.49 et_16.50 al_16.51._16.52 1997_16.53;_16.54 Derkatch_16.55 et_16.56 al_16.57._16.58 2001_16.59)_16.60._16.61

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).

Aggregates_17.1 of_17.2 the_17.3 New1p_17.4 prion_17.5 domain_17.6,_17.7 whether_17.8 resulting_17.9 from_17.10 overexpression_17.11 or_17.12 from_17.13 a_17.14 constitutive_17.15 prion_17.16 form_17.17 (_17.18termed_17.19 [_17.20NU_17.21+_17.22]_17.23)_17.24,_17.25 also_17.26 promoted_17.27 the_17.28 aggregation_17.29 of_17.30 other_17.31 Q/N-rich_17.32 proteins_17.33,_17.34 emulating_17.35 the_17.36 effect_17.37 of_17.38 [_17.39PIN_17.40+_17.41]_17.42 (_17.43Osherovich_17.44 and_17.45 Weissman_17.46 2001_17.47)_17.48._17.49

Figure 1.

Figure_18.1 1_18.2._18.3

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.

Schematic_19.1 Diagram_19.2 of_19.3 Sup35p_19.4 and_19.5 New1pPrion_19.6 domains_19.7 of_19.8 both_19.9 proteins_19.10 are_19.11 enlarged_19.12 in_19.13 the_19.14 center_19.15,_19.16 highlighting_19.17 the_19.18 Q/N-rich_19.19 tract_19.20 of_19.21 Sup35p_19.22 (_19.23blue_19.24)_19.25,_19.26 the_19.27 NYN_19.28 tripeptide_19.29 repeat_19.30 of_19.31 New1p_19.32 (_19.33purple_19.34)_19.35,_19.36 and_19.37 the_19.38 oligopeptide_19.39 repeat_19.40 sequences_19.41 (_19.42orange_19.43)_19.44 found_19.45 in_19.46 both_19.47 proteins_19.48._19.49

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.

The_20.1 sequence_20.2 of_20.3 the_20.4 NEW1_20.5 oligopetide_20.6 repeat_20.7 (_20.8residues_20.9 50_20.10–_20.1170_20.12)_20.13 is_20.14 QQQRNWKQGGNYQQGGYQSYN_20.15,_20.16 while_20.17 that_20.18 of_20.19 the_20.20 adjacent_20.21 tripeptide_20.22 repeat_20.23 region_20.24 (_20.25residues_20.26 71_20.27–_20.28100_20.29)_20.30 is_20.31 SNYNNYNNYNNYNNYNNYNNYNKYNGQGYQ_20.32._20.33

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).

Many_21.1 sequences_21.2 with_21.3 Q/N_21.4 content_21.5 as_21.6 high_21.7 as_21.8 that_21.9 of_21.10 Sup35p_21.11 and_21.12 Ure2p_21.13,_21.14 including_21.15 human_21.16 polyglutamine_21.17 expansion_21.18 disease_21.19 proteins_21.20,_21.21 form_21.22 visible_21.23 aggregates_21.24 when_21.25 overexpressed_21.26 in_21.27 yeast_21.28 as_21.29 GFP_21.30 fusions_21.31 (_21.32Krobitsch_21.33 and_21.34 Lindquist_21.35 2000_21.36;_21.37 Osherovich_21.38 and_21.39 Weissman_21.40 2001_21.41;_21.42 Meriin_21.43 et_21.44 al_21.45._21.46 2002_21.47)_21.48._21.49

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).

However_22.1,_22.2 only_22.3 a_22.4 limited_22.5 number_22.6 of_22.7 Q/N-rich_22.8 sequences_22.9 are_22.10 bone_22.11 fide_22.12 prion_22.13 domains_22.14 capable_22.15 of_22.16 propagating_22.17 these_22.18 aggregates_22.19 over_22.20 multiple_22.21 cell_22.22 generations_22.23 even_22.24 when_22.25 expressed_22.26 at_22.27 low_22.28 levels_22.29 (_22.30J_22.31._22.32 Hood_22.33 and_22.34 J_22.35._22.36S_22.37._22.38W_22.39,_22.40 unpublished_22.41 data_22.42)_22.43._22.44

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.

To_23.1 understand_23.2 what_23.3 distinguishes_23.4 generic_23.5 Q/N-rich_23.6 aggregates_23.7 from_23.8 heritable_23.9 prions_23.10,_23.11 we_23.12 conducted_23.13 a_23.14 detailed_23.15 dissection_23.16 of_23.17 the_23.18 prion-forming_23.19 regions_23.20 of_23.21 Sup35p_23.22 and_23.23 New1p_23.24._23.25

We found that the prion properties of Sup35p and New1p require the presence of two independent and portable sequence elements within their prion domains.

We_24.1 found_24.2 that_24.3 the_24.4 prion_24.5 properties_24.6 of_24.7 Sup35p_24.8 and_24.9 New1p_24.10 require_24.11 the_24.12 presence_24.13 of_24.14 two_24.15 independent_24.16 and_24.17 portable_24.18 sequence_24.19 elements_24.20 within_24.21 their_24.22 prion_24.23 domains_24.24._24.25

One element mediates the growth of prion aggregates by incorporation of soluble monomers.

One_25.1 element_25.2 mediates_25.3 the_25.4 growth_25.5 of_25.6 prion_25.7 aggregates_25.8 by_25.9 incorporation_25.10 of_25.11 soluble_25.12 monomers_25.13._25.14

The second promotes the inheritance of aggregates, generating new heritable “seeds” which can be partitioned between mother and daughter cells during cell division.

The_26.1 second_26.2 promotes_26.3 the_26.4 inheritance_26.5 of_26.6 aggregates_26.7,_26.8 generating_26.9 new_26.10 heritable_26.11 “_26.12seeds_26.13”_26.14 which_26.15 can_26.16 be_26.17 partitioned_26.18 between_26.19 mother_26.20 and_26.21 daughter_26.22 cells_26.23 during_26.24 cell_26.25 division_26.26._26.27

Results

Results_27.1

Distinct Regions of the New1p Prion Domain Mediate Prion Growth and Division

Distinct_28.1 Regions_28.2 of_28.3 the_28.4 New1p_28.5 Prion_28.6 Domain_28.7 Mediate_28.8 Prion_28.9 Growth_28.10 and_28.11 Division_28.12

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).

Sup35p_29.1 can_29.2 alternate_29.3 between_29.4 a_29.5 biochemically_29.6 active_29.7,_29.8 soluble_29.9 form_29.10 (_29.11[_29.12psi_29.13–_29.14]_29.15)_29.16 and_29.17 an_29.18 aggregated_29.19 prion_29.20 state_29.21 (_29.22[_29.23PSI_29.24+_29.25]_29.26)_29.27 with_29.28 diminished_29.29 translation_29.30 termination_29.31 activity_29.32,_29.33 which_29.34 can_29.35 be_29.36 monitored_29.37 by_29.38 nonsense_29.39 suppression_29.40 of_29.41 the_29.42 mutant_29.43 ade1_29.44–_29.4514_29.46 allele_29.47 (_29.48Liebman_29.49 and_29.50 Derkatch_29.51 1999_29.52)_29.53._29.54

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+).

Whereas_30.1 [_30.2psi_30.3–_30.4]_30.5 strains_30.6 form_30.7 red_30.8 colonies_30.9 on_30.10 yeast_30.11 extract-peptone-dextrose_30.12 (_30.13YEPD_30.14)_30.15 medium_30.16 and_30.17 cannot_30.18 grow_30.19 in_30.20 the_30.21 absence_30.22 of_30.23 adenine_30.24,_30.25 [_30.26PSI_30.27+_30.28]_30.29 strains_30.30 suppress_30.31 the_30.32 premature_30.33 stop_30.34 codon_30.35 in_30.36 ade1-14_30.37,_30.38 and_30.39 thus_30.40 appear_30.41 pink_30.42 or_30.43 white_30.44 on_30.45 YEPD_30.46 medium_30.47 and_30.48 grow_30.49 on_30.50 adenine-free_30.51 medium_30.52 (_30.53a_30.54 phenotype_30.55 termed_30.56 adenine_30.57 prototrophy_30.58,_30.59 Ade_30.60+_30.61)_30.62._30.63

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).

The_31.1 N_31.2 or_31.3 prion_31.4 domain_31.5 of_31.6 Sup35p_31.7 (_31.8residues_31.9 1-112_31.10)_31.11 is_31.12 required_31.13 for_31.14 [_31.15PSI_31.16+_31.17]_31.18 formation_31.19 but_31.20 is_31.21 dispensable_31.22 for_31.23 the_31.24 translation_31.25 termination_31.26 activity_31.27 of_31.28 the_31.29 carboxy-terminal_31.30 C_31.31 domain_31.32 (_31.33Ter-Avanesyan_31.34 et_31.35 al_31.36._31.37 1993_31.38)_31.39._31.40

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).

The_32.1 charged_32.2 middle_32.3 domain_32.4 (_32.5M_32.6)_32.7 is_32.8 not_32.9 required_32.10 for_32.11 prion_32.12 behavior_32.13,_32.14 but_32.15 modulates_32.16 the_32.17 efficiency_32.18 of_32.19 chaperone-dependent_32.20 prion_32.21 transmission_32.22 (_32.23Liu_32.24 et_32.25 al_32.26._32.27 2002_32.28;_32.29 L_32.30._32.31Z_32.32._32.33O_32.34._32.35,_32.36 unpublished_32.37 data_32.38)_32.39 (_32.40Figure_32.41 1_32.42)_32.43._32.44

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).

Two_33.1 distinct_33.2 regions_33.3 in_33.4 the_33.5 N_33.6 domain_33.7 have_33.8 previously_33.9 been_33.10 implicated_33.11 in_33.12 Sup35p_33.13 aggregation_33.14:_33.15 a_33.16 Q/N-rich_33.17 tract_33.18 (_33.19residues_33.20 1_33.21–_33.2239_33.23)_33.24 (_33.25DePace_33.26 et_33.27 al_33.28._33.29 1998_33.30)_33.31 and_33.32 an_33.33 oligopeptide_33.34 repeat_33.35 (_33.36residues_33.37 40_33.38–_33.39112_33.40)_33.41 that_33.42 consists_33.43 of_33.44 five_33.45 and_33.46 a_33.47 half_33.48 degenerate_33.49 repeats_33.50 of_33.51 the_33.52 consensus_33.53 sequence_33.54 P/QQGGYQQ/SYN_33.55 (_33.56Liu_33.57 and_33.58 Lindquist_33.59 1999_33.60;_33.61 Parham_33.62 et_33.63 al_33.64._33.65 2001_33.66;_33.67 Crist_33.68 et_33.69 al_33.70._33.71 2003_33.72)_33.73._33.74

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).

We_34.1 had_34.2 earlier_34.3 identified_34.4 New1p_34.5 as_34.6 an_34.7 uncharacterized_34.8 protein_34.9 with_34.10 a_34.11 Sup35p-like_34.12 N-terminal_34.13 domain_34.14;_34.15 when_34.16 fused_34.17 to_34.18 the_34.19 M_34.20 and_34.21 C_34.22 domains_34.23 of_34.24 Sup35p_34.25,_34.26 the_34.27 first_34.28 153_34.29 residues_34.30 of_34.31 New1p_34.32 (_34.33New11_34.34–_34.35153_34.36)_34.37 supported_34.38 a_34.39 [_34.40PSI_34.41+_34.42]_34.43-like_34.44 prion_34.45 state_34.46 termed_34.47 [_34.48NU_34.49+_34.50]_34.51 (_34.52Santoso_34.53 et_34.54 al_34.55._34.56 2000_34.57)_34.58._34.59

Sup35p and New1p have regions of clear similarity beyond their high Q/N content (Figure 1).

Sup35p_35.1 and_35.2 New1p_35.3 have_35.4 regions_35.5 of_35.6 clear_35.7 similarity_35.8 beyond_35.9 their_35.10 high_35.11 Q/N_35.12 content_35.13 (_35.14Figure_35.15 1_35.16)_35.17._35.18

The prion domains of both have Q/N-rich tracts and oligopeptide repeat regions, although their order is reversed.

The_36.1 prion_36.2 domains_36.3 of_36.4 both_36.5 have_36.6 Q/N-rich_36.7 tracts_36.8 and_36.9 oligopeptide_36.10 repeat_36.11 regions_36.12,_36.13 although_36.14 their_36.15 order_36.16 is_36.17 reversed_36.18._36.19

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).

The_37.1 C-terminal_37.2 domains_37.3 of_37.4 New1p_37.5 and_37.6 Sup35p_37.7 also_37.8 appear_37.9 to_37.10 be_37.11 related_37.12,_37.13 based_37.14 on_37.15 modest_37.16 homology_37.17 and_37.18 the_37.19 similarity_37.20 of_37.21 the_37.22 translation_37.23 termination_37.24 defects_37.25 in_37.26 sup35_37.27 (_37.28Song_37.29 and_37.30 Liebman_37.31 1985_37.32)_37.33 and_37.34 new1_37.35 mutants_37.36 (_37.37L_37.38._37.39Z_37.40._37.41O_37.42._37.43,_37.44 unpublished_37.45 data_37.46)_37.47._37.48

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.

To_38.1 understand_38.2 the_38.3 sequence_38.4 requirements_38.5 for_38.6 the_38.7 prion_38.8 behavior_38.9 of_38.10 New1p_38.11,_38.12 we_38.13 constructed_38.14 a_38.15 series_38.16 of_38.17 truncated_38.18 prion_38.19 domains_38.20 (_38.21Figure_38.22 2A_38.23)_38.24 and_38.25 examined_38.26 their_38.27 participation_38.28 in_38.29 several_38.30 critical_38.31 steps_38.32 of_38.33 the_38.34 prion_38.35 replication_38.36 cycle_38.37._38.38

We first asked whether these truncated prion domains could form visible foci when fused to GFP (aggregation).

We_39.1 first_39.2 asked_39.3 whether_39.4 these_39.5 truncated_39.6 prion_39.7 domains_39.8 could_39.9 form_39.10 visible_39.11 foci_39.12 when_39.13 fused_39.14 to_39.15 GFP_39.16 (_39.17aggregation_39.18)_39.19._39.20

Next, we examined whether such aggregates could convert New11–153 into a [NU+] prion state (induction).

Next_40.1,_40.2 we_40.3 examined_40.4 whether_40.5 such_40.6 aggregates_40.7 could_40.8 convert_40.9 New11_40.10–_40.11153_40.12 into_40.13 a_40.14 [_40.15NU_40.16+_40.17]_40.18 prion_40.19 state_40.20 (_40.21induction_40.22)_40.23._40.24

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).

Finally_41.1,_41.2 we_41.3 fused_41.4 these_41.5 constructs_41.6 to_41.7 the_41.8 M_41.9 and_41.10 C_41.11 domains_41.12 of_41.13 Sup35p_41.14 (_41.15–_41.16M-C_41.17)_41.18,_41.19 introduced_41.20 them_41.21 in_41.22 place_41.23 of_41.24 endogenous_41.25 SUP35_41.26,_41.27 and_41.28 assessed_41.29 whether_41.30 these_41.31 proteins_41.32 could_41.33 adopt_41.34 stable_41.35 prion_41.36 states_41.37 (_41.38maintenance_41.39)_41.40._41.41

We found that distinct regions within the New1p prion domain are necessary for the induction and maintenance of [NU+] (Figure 2A).

We_42.1 found_42.2 that_42.3 distinct_42.4 regions_42.5 within_42.6 the_42.7 New1p_42.8 prion_42.9 domain_42.10 are_42.11 necessary_42.12 for_42.13 the_42.14 induction_42.15 and_42.16 maintenance_42.17 of_42.18 [_42.19NU_42.20+_42.21]_42.22 (_42.23Figure_42.24 2A_42.25)_42.26._42.27

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+].

The_43.1 asparagine-tyrosine-asparagine_43.2 (_43.3NYN_43.4)_43.5 repeat_43.6 (_43.7residues_43.8 70_43.9–_43.10100_43.11)_43.12,_43.13 which_43.14 we_43.15 had_43.16 earlier_43.17 shown_43.18 to_43.19 be_43.20 sufficient_43.21 for_43.22 aggregation_43.23 (_43.24Osherovich_43.25 and_43.26 Weissman_43.27 2001_43.28)_43.29,_43.30 also_43.31 proved_43.32 sufficient_43.33 for_43.34 induction_43.35 of_43.36 [_43.37NU_43.38+_43.39]_43.40._43.41

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).

As_44.1 with_44.2 the_44.3 full-length_44.4 New1p_44.5 prion_44.6 domain_44.7,_44.8 overexpression_44.9 of_44.10 the_44.11 NYN_44.12 repeat_44.13 efficiently_44.14 stimulated_44.15 the_44.16 appearance_44.17 of_44.18 Ade_44.19+_44.20 in_44.21 [_44.22nu_44.23–_44.24]_44.25 cells_44.26 (_44.27Figure_44.28 2B_44.29,_44.30 left_44.31)_44.32._44.33

However, stable prion maintenance required both the NYN repeat and the adjacent oligopeptide repeat.

However_45.1,_45.2 stable_45.3 prion_45.4 maintenance_45.5 required_45.6 both_45.7 the_45.8 NYN_45.9 repeat_45.10 and_45.11 the_45.12 adjacent_45.13 oligopeptide_45.14 repeat_45.15._45.16

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).

In_46.1 a_46.2 strain_46.3 with_46.4 this_46.5 minimized_46.6 New1p_46.7 prion_46.8 domain_46.9 (_46.10residues_46.11 50_46.12–_46.13100_46.14)_46.15,_46.16 overexpression_46.17 of_46.18 the_46.19 full_46.20 prion_46.21 domain_46.22 or_46.23 of_46.24 the_46.25 NYN_46.26 repeat_46.27 alone_46.28 promoted_46.29 the_46.30 appearance_46.31 of_46.32 Ade_46.33+_46.34 colonies_46.35 (_46.36Figure_46.37 2B_46.38,_46.39 right_46.40)_46.41._46.42

The resulting convertants remained Ade+ after loss of the inducer plasmid but reverted to Ade- after transient GuHCl treatment (Figure 2C).

The_47.1 resulting_47.2 convertants_47.3 remained_47.4 Ade_47.5+_47.6 after_47.7 loss_47.8 of_47.9 the_47.10 inducer_47.11 plasmid_47.12 but_47.13 reverted_47.14 to_47.15 Ade- after_47.16 transient_47.17 GuHCl_47.18 treatment_47.19 (_47.20Figure_47.21 2C_47.22)_47.23._47.24

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+].

We_48.1 conclude_48.2 that_48.3 the_48.4 oligopeptide_48.5 repeat_48.6 and_48.7 the_48.8 NYN_48.9 repeat_48.10 of_48.11 New1p_48.12 together_48.13 are_48.14 sufficient_48.15 to_48.16 support_48.17 a_48.18 prion_48.19 state_48.20,_48.21 termed_48.22 [_48.23NU_48.24+_48.25]_48.26mini_48.27,_48.28 which_48.29 recapitulates_48.30 the_48.31 characteristics_48.32 of_48.33 [_48.34NU_48.35+_48.36]_48.37._48.38

Figure 2.

Figure_49.1 2_49.2._49.3

Dissection of the New1p Prion Domain Reveals Distinct Regions Responsible for Aggregation and Prion Inheritance

Dissection_50.1 of_50.2 the_50.3 New1p_50.4 Prion_50.5 Domain_50.6 Reveals_50.7 Distinct_50.8 Regions_50.9 Responsible_50.10 for_50.11 Aggregation_50.12 and_50.13 Prion_50.14 Inheritance_50.15

(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+].

(_51.1A_51.2)_51.3 Indicated_51.4 fragments_51.5 of_51.6 New1p_51.7 (_51.8left_51.9)_51.10 were_51.11 expressed_51.12 as_51.13 GFP_51.14 fusions_51.15 (_51.16inducers_51.17)_51.18 in_51.19 a_51.20 [_51.21nu_51.22–_51.23]_51.24 [_51.25pin_51.26–_51.27]_51.28 strain_51.29,_51.30 examined_51.31 by_51.32 microscopy_51.33 for_51.34 GFP_51.35 aggregation_51.36,_51.37 then_51.38 plated_51.39 on_51.40 SD-ade_51.41 medium_51.42 to_51.43 assess_51.44 induction_51.45 of_51.46 [_51.47NU_51.48+_51.49]_51.50._51.51

The symbol “+” indicates induction frequencies of at least 5%; the symbol “–” indicates no induction.

The_52.1 symbol_52.2 “_52.3+_52.4”_52.5 indicates_52.6 induction_52.7 frequencies_52.8 of_52.9 at_52.10 least_52.11 5%;_52.12 the_52.13 symbol_52.14 “_52.15–_52.16”_52.17 indicates_52.18 no_52.19 induction_52.20._52.21

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).

Maintenance_53.1 was_53.2 assessed_53.3 by_53.4 the_53.5 ability_53.6 of_53.7 an_53.8 episomal_53.9 maintainer_53.10 version_53.11 of_53.12 the_53.13 indicated_53.14 fragment_53.15 to_53.16 support_53.17 an_53.18 Ade_53.19+_53.20 state_53.21 after_53.22 overexpression_53.23 of_53.24 New11_53.25–_53.26153-GFP_53.27 (_53.28see_53.29 Materials_53.30 and_53.31 Methods_53.32)_53.33._53.34

The aggregation of New1-GFP fusions (second column) has been previously reported (Osherovich and Weissman 2001).

The_54.1 aggregation_54.2 of_54.3 New1-GFP_54.4 fusions_54.5 (_54.6second_54.7 column_54.8)_54.9 has_54.10 been_54.11 previously_54.12 reported_54.13 (_54.14Osherovich_54.15 and_54.16 Weissman_54.17 2001_54.18)_54.19._54.20

(B) The NYN repeat of New1p induces [NU+] and [NU+]mini.

(_55.1B_55.2)_55.3 The_55.4 NYN_55.5 repeat_55.6 of_55.7 New1p_55.8 induces_55.9 [_55.10NU_55.11+_55.12]_55.13 and_55.14 [_55.15NU_55.16+_55.17]_55.18mini_55.19._55.20

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.

New170_56.1–_56.2100-GFP_56.3 was_56.4 overexpressed_56.5 in_56.6 [_56.7nu_56.8–_56.9]_56.10 and_56.11 [_56.12nu_56.13–_56.14]_56.15mini_56.16 strains_56.17 (_56.18[_56.19pin_56.20–_56.21]_56.22 and_56.23 [_56.24PIN_56.25+_56.26]_56.27 derivatives_56.28 of_56.29 each_56.30)_56.31,_56.32 along_56.33 with_56.34 vector_56.35 only_56.36 or_56.37 New11_56.38–_56.39153-GFP_56.40 controls_56.41._56.42

Averages of three independent trials, representing 600–2000 colonies, are shown for most induction experiments; inductions using New170–100-GFP were conducted twice.

Averages_57.1 of_57.2 three_57.3 independent_57.4 trials_57.5,_57.6 representing_57.7 600_57.8–_57.92000_57.10 colonies_57.11,_57.12 are_57.13 shown_57.14 for_57.15 most_57.16 induction_57.17 experiments_57.18;_57.19 inductions_57.20 using_57.21 New170_57.22–_57.23100-GFP_57.24 were_57.25 conducted_57.26 twice_57.27._57.28

Error bars show minimal and maximal observed induction efficiencies.

Error_58.1 bars_58.2 show_58.3 minimal_58.4 and_58.5 maximal_58.6 observed_58.7 induction_58.8 efficiencies_58.9._58.10

(C) Reversibility of [NU+]mini.

(_59.1C_59.2)_59.3 Reversibility_59.4 of_59.5 [_59.6NU_59.7+_59.8]_59.9mini_59.10._59.11

The [pin–] Ade+ convertants obtained in (B) were colony purified on SD-ade medium and confirmed to have lost the inducer plasmid.

The_60.1 [_60.2pin_60.3–_60.4]_60.5 Ade_60.6+_60.7 convertants_60.8 obtained_60.9 in_60.10 (_60.11B_60.12)_60.13 were_60.14 colony_60.15 purified_60.16 on_60.17 SD-ade_60.18 medium_60.19 and_60.20 confirmed_60.21 to_60.22 have_60.23 lost_60.24 the_60.25 inducer_60.26 plasmid_60.27._60.28

A stable [NU+]mini isolate is shown before and after induction, as well as after GuHCl treatment, along with [nu–] and [NU+] reference strains.

A_61.1 stable_61.2 [_61.3NU_61.4+_61.5]_61.6mini_61.7 isolate_61.8 is_61.9 shown_61.10 before_61.11 and_61.12 after_61.13 induction_61.14,_61.15 as_61.16 well_61.17 as_61.18 after_61.19 GuHCl_61.20 treatment_61.21,_61.22 along_61.23 with_61.24 [_61.25nu_61.26–_61.27]_61.28 and_61.29 [_61.30NU_61.31+_61.32]_61.33 reference_61.34 strains_61.35._61.36

Dissection of the Sup35p Prion Domain

Dissection_62.1 of_62.2 the_62.3 Sup35p_62.4 Prion_62.5 Domain_62.6

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).

In_63.1 light_63.2 of_63.3 the_63.4 similarity_63.5 between_63.6 New1p_63.7 and_63.8 Sup35p_63.9 prion_63.10 domains_63.11,_63.12 we_63.13 asked_63.14 whether_63.15 separate_63.16 regions_63.17 of_63.18 Sup35p_63.19 were_63.20 involved_63.21 in_63.22 the_63.23 induction_63.24 and_63.25 maintenance_63.26 of_63.27 [_63.28PSI_63.29+_63.30]_63.31 aggregates_63.32 (_63.33Figure_63.34 3_63.35)_63.36._63.37

We constructed a series of truncated Sup35p N domains and analyzed their behavior in the aggregation, induction, and maintenance assays described above for [NU+].

We_64.1 constructed_64.2 a_64.3 series_64.4 of_64.5 truncated_64.6 Sup35p_64.7 N_64.8 domains_64.9 and_64.10 analyzed_64.11 their_64.12 behavior_64.13 in_64.14 the_64.15 aggregation_64.16,_64.17 induction_64.18,_64.19 and_64.20 maintenance_64.21 assays_64.22 described_64.23 above_64.24 for_64.25 [_64.26NU_64.27+_64.28]_64.29._64.30

Additionally, we examined the ability of truncated N domains to decorate preexisting Sup35p aggregates in [PSI+] strains.

Additionally_65.1,_65.2 we_65.3 examined_65.4 the_65.5 ability_65.6 of_65.7 truncated_65.8 N_65.9 domains_65.10 to_65.11 decorate_65.12 preexisting_65.13 Sup35p_65.14 aggregates_65.15 in_65.16 [_65.17PSI_65.18+_65.19]_65.20 strains_65.21._65.22

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.

We_66.1 found_66.2 that_66.3 the_66.4 Q/N-rich_66.5 tract_66.6 and_66.7 a_66.8 small_66.9 portion_66.10 of_66.11 the_66.12 adjacent_66.13 oligopeptide_66.14 repeat_66.15 are_66.16 responsible_66.17 for_66.18 Sup35p_66.19 aggregation_66.20 and_66.21 de_66.22 novo_66.23 [_66.24PSI_66.25+_66.26]_66.27 induction_66.28._66.29

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).

Deletions_67.1 within_67.2 the_67.3 Q/N-rich_67.4 tract_67.5 or_67.6 of_67.7 oligopeptide_67.8 repeat_67.9 1_67.10 abolished_67.11 these_67.12 properties_67.13,_67.14 whereas_67.15 a_67.16 construct_67.17 containing_67.18 only_67.19 the_67.20 Q/N-rich_67.21 region_67.22 and_67.23 the_67.24 first_67.25 two_67.26 oligopeptide_67.27 repeats_67.28 (_67.29residues_67.30 1_67.31–_67.3264_67.33)_67.34 aggregated_67.35 and_67.36 induced_67.37 [_67.38PSI_67.39+_67.40]_67.41 at_67.42 levels_67.43 comparable_67.44 to_67.45 the_67.46 full_67.47 prion_67.48 domain_67.49,_67.50 in_67.51 agreement_67.52 with_67.53 King_67.54 (_67.552001_67.56)_67.57._67.58

A construct (residues 1–57) with a partial deletion of oligopeptide repeat 2 could still aggregate and induce [PSI+], albeit with decreased efficiency.

A_68.1 construct_68.2 (_68.3residues_68.4 1_68.5–_68.657_68.7)_68.8 with_68.9 a_68.10 partial_68.11 deletion_68.12 of_68.13 oligopeptide_68.14 repeat_68.15 2_68.16 could_68.17 still_68.18 aggregate_68.19 and_68.20 induce_68.21 [_68.22PSI_68.23+_68.24]_68.25,_68.26 albeit_68.27 with_68.28 decreased_68.29 efficiency_68.30._68.31

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.

Although_69.1 a_69.2 construct_69.3 lacking_69.4 oligopeptide_69.5 repeat_69.6 2_69.7 entirely_69.8 (_69.9residues_69.10 1_69.11–_69.1249_69.13)_69.14 did_69.15 not_69.16 induce_69.17 [_69.18PSI_69.19+_69.20]_69.21 de_69.22 novo_69.23,_69.24 this_69.25 GFP_69.26 fusion_69.27 could_69.28 nonetheless_69.29 decorate_69.30 preexisting_69.31 Sup35p_69.32 aggregates_69.33._69.34

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.

Thus_70.1,_70.2 while_70.3 oligopeptide_70.4 repeat_70.5 2_70.6 contributes_70.7 to_70.8 the_70.9 aggregation_70.10 of_70.11 Sup35p_70.12,_70.13 the_70.14 primary_70.15 determinants_70.16 of_70.17 prion_70.18 induction_70.19 reside_70.20 in_70.21 the_70.22 amino-terminal_70.23 Q/N-rich_70.24 region_70.25 and_70.26 oligopeptide_70.27 repeat_70.28 1_70.29._70.30

In contrast, the rest of the oligopeptide repeat region is needed for stable inheritance of [PSI+] aggregates.

In_71.1 contrast_71.2,_71.3 the_71.4 rest_71.5 of_71.6 the_71.7 oligopeptide_71.8 repeat_71.9 region_71.10 is_71.11 needed_71.12 for_71.13 stable_71.14 inheritance_71.15 of_71.16 [_71.17PSI_71.18+_71.19]_71.20 aggregates_71.21._71.22

Constructs that did not form fluorescent foci could not retain [PSI+], suggesting that aggregation is a prerequisite for prion maintenance.

Constructs_72.1 that_72.2 did_72.3 not_72.4 form_72.5 fluorescent_72.6 foci_72.7 could_72.8 not_72.9 retain_72.10 [_72.11PSI_72.12+_72.13]_72.14,_72.15 suggesting_72.16 that_72.17 aggregation_72.18 is_72.19 a_72.20 prerequisite_72.21 for_72.22 prion_72.23 maintenance_72.24._72.25

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+].

However_73.1,_73.2 aggregation_73.3 is_73.4 not_73.5 sufficient_73.6 for_73.7 prion_73.8 inheritance_73.9,_73.10 as_73.11 Sup35p_73.12 constructs_73.13 with_73.14 deletions_73.15 spanning_73.16 oligopeptide_73.17 repeats_73.18 3_73.19–_73.205_73.21 could_73.22 not_73.23 support_73.24 a_73.25 prion_73.26 state_73.27 despite_73.28 their_73.29 ability_73.30 to_73.31 form_73.32 aggregates_73.33 and_73.34 efficiently_73.35 induce_73.36 [_73.37PSI_73.38+_73.39]_73.40._73.41

Only the sixth (incomplete) oligopeptide repeat proved dispensable for [PSI+] maintenance, consistent with an earlier report (Parham et al. 2001).

Only_74.1 the_74.2 sixth_74.3 (_74.4incomplete_74.5)_74.6 oligopeptide_74.7 repeat_74.8 proved_74.9 dispensable_74.10 for_74.11 [_74.12PSI_74.13+_74.14]_74.15 maintenance_74.16,_74.17 consistent_74.18 with_74.19 an_74.20 earlier_74.21 report_74.22 (_74.23Parham_74.24 et_74.25 al_74.26._74.27 2001_74.28)_74.29._74.30

The PNM2-1 Mutation in Oligopeptide Repeat 2 Specifically Compromises the Inheritance of [PSI+]

The_75.1 PNM2-1_75.2 Mutation_75.3 in_75.4 Oligopeptide_75.5 Repeat_75.6 2_75.7 Specifically_75.8 Compromises_75.9 the_75.10 Inheritance_75.11 of_75.12 [_75.13PSI_75.14+_75.15]_75.16

Our deletion analysis suggested that oligopeptide repeat 2 participated in both the formation and inheritance of Sup35p aggregates.

Our_76.1 deletion_76.2 analysis_76.3 suggested_76.4 that_76.5 oligopeptide_76.6 repeat_76.7 2_76.8 participated_76.9 in_76.10 both_76.11 the_76.12 formation_76.13 and_76.14 inheritance_76.15 of_76.16 Sup35p_76.17 aggregates_76.18._76.19

We made use of a point mutation within oligopeptide repeat 2 known as PNM2-1 (G58D) to distinguish between these two functions.

We_77.1 made_77.2 use_77.3 of_77.4 a_77.5 point_77.6 mutation_77.7 within_77.8 oligopeptide_77.9 repeat_77.10 2_77.11 known_77.12 as_77.13 PNM2-1_77.14 (_77.15G58D_77.16)_77.17 to_77.18 distinguish_77.19 between_77.20 these_77.21 two_77.22 functions_77.23._77.24

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).

PNM2-1_78.1 (_78.2PSI_78.3 No_78.4 More_78.5)_78.6 shows_78.7 strong_78.8 interference_78.9 with_78.10 [_78.11PSI_78.12+_78.13]_78.14 in_78.15 certain_78.16 strain_78.17 backgrounds_78.18 through_78.19 a_78.20 poorly_78.21 understood_78.22 mechanism_78.23 (_78.24McCready_78.25 et_78.26 al_78.27._78.28 1977_78.29;_78.30 Doel_78.31 et_78.32 al_78.33._78.34 1994_78.35;_78.36 Kochneva-Pervukhova_78.37 et_78.38 al_78.39._78.40 1998_78.41;_78.42 Derkatch_78.43 et_78.44 al_78.45._78.46 1999_78.47)_78.48._78.49

Using both in vivo and in vitro assays, we established that PNM2-1 does not have a defect in aggregation or [PSI+] induction.

Using_79.1 both_79.2 in_79.3 vivo_79.4 and_79.5 in_79.6 vitro_79.7 assays_79.8,_79.9 we_79.10 established_79.11 that_79.12 PNM2-1_79.13 does_79.14 not_79.15 have_79.16 a_79.17 defect_79.18 in_79.19 aggregation_79.20 or_79.21 [_79.22PSI_79.23+_79.24]_79.25 induction_79.26._79.27

Earlier work indicated that PNM2-1 is capable of seeding [PSI+] in vivo (Kochneva-Pervukhova et al. 1998; Derkatch et al. 1999; King 2001).

Earlier_80.1 work_80.2 indicated_80.3 that_80.4 PNM2-1_80.5 is_80.6 capable_80.7 of_80.8 seeding_80.9 [_80.10PSI_80.11+_80.12]_80.13 in_80.14 vivo_80.15 (_80.16Kochneva-Pervukhova_80.17 et_80.18 al_80.19._80.20 1998_80.21;_80.22 Derkatch_80.23 et_80.24 al_80.25._80.26 1999_80.27;_80.28 King_80.29 2001_80.30)_80.31._80.32

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).

Consistent_81.1 with_81.2 these_81.3 reports_81.4,_81.5 we_81.6 found_81.7 that_81.8 overexpression_81.9 of_81.10 a_81.11 PNM2-1-GFP_81.12 fusion_81.13 in_81.14 [_81.15psi_81.16–_81.17]_81.18 [_81.19PIN_81.20+_81.21]_81.22 cells_81.23 with_81.24 a_81.25 wild-type_81.26 SUP35_81.27 locus_81.28 led_81.29 to_81.30 both_81.31 focus_81.32 formation_81.33 and_81.34 [_81.35PSI_81.36+_81.37]_81.38 induction_81.39 (_81.40Figure_81.41 4A_81.42)_81.43._81.44

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).

A_82.1 previous_82.2 study_82.3 of_82.4 Sup35p_82.5 polymerization_82.6 in_82.7 extracts_82.8 had_82.9 suggested_82.10 that_82.11 PNM2-1_82.12 might_82.13 interfere_82.14 with_82.15 [_82.16PSI_82.17+_82.18]_82.19 through_82.20 a_82.21 defect_82.22 in_82.23 seeding_82.24 (_82.25Kochneva-Pervukhova_82.26 et_82.27 al_82.28._82.29 1998_82.30)_82.31._82.32

We tested this by examining the rate of seeded polymerization of recombinant PNM2-1 protein.

We_83.1 tested_83.2 this_83.3 by_83.4 examining_83.5 the_83.6 rate_83.7 of_83.8 seeded_83.9 polymerization_83.10 of_83.11 recombinant_83.12 PNM2-1_83.13 protein_83.14._83.15

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).

Like_84.1 wild-type_84.2 Sup35p_84.3,_84.4 purified_84.5 PNM2-1_84.6 spontaneously_84.7 formed_84.8 amyloid_84.9 fibrils_84.10 in_84.11 vitro_84.12;_84.13 this_84.14 was_84.15 accelerated_84.16 by_84.17 the_84.18 addition_84.19 of_84.20 preformed_84.21 Sup35p_84.22 polymer_84.23 seeds_84.24 (_84.25data_84.26 not_84.27 shown_84.28)_84.29._84.30

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.

We_85.1 measured_85.2 the_85.3 initial_85.4 rates_85.5 of_85.6 polymerization_85.7 of_85.8 wild-type_85.9 and_85.10 PNM2-1_85.11 protein_85.12 seeded_85.13 by_85.14 preformed_85.15 wild-type_85.16 polymers_85.17 (_85.18Figure_85.19 4B_85.20)_85.21 and_85.22 by_85.23 PNM2-1_85.24 polymers_85.25 (_85.26Figure_85.27 4C_85.28)_85.29 using_85.30 a_85.31 thioflavin-T_85.32–_85.33binding_85.34 assay_85.35._85.36

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.

We_86.1 observed_86.2 that_86.3 wild-type_86.4 and_86.5 PNM2-1_86.6 monomers_86.7 were_86.8 seeded_86.9 by_86.10 wild-type_86.11 polymers_86.12 with_86.13 similar_86.14 kinetics_86.15;_86.16 likewise_86.17,_86.18 PNM2-1_86.19 polymers_86.20 seeded_86.21 both_86.22 wild-type_86.23 and_86.24 PNM2-1_86.25 monomers_86.26 equivalently_86.27._86.28

Thus, the PNM2-1 mutation does not affect polymerization or seeding.

Thus_87.1,_87.2 the_87.3 PNM2-1_87.4 mutation_87.5 does_87.6 not_87.7 affect_87.8 polymerization_87.9 or_87.10 seeding_87.11._87.12

Figure 3.

Figure_88.1 3_88.2._88.3

Dissection of the Sup35p Prion Domain

Dissection_89.1 of_89.2 the_89.3 Sup35p_89.4 Prion_89.5 Domain_89.6

At top are schematic diagrams of these experiments; positive outcomes are shown below the arrows.

At_90.1 top_90.2 are_90.3 schematic_90.4 diagrams_90.5 of_90.6 these_90.7 experiments_90.8;_90.9 positive_90.10 outcomes_90.11 are_90.12 shown_90.13 below_90.14 the_90.15 arrows_90.16._90.17

In some cases, similar experiments have been reported by Parham et al. (2001) (indicated by “a”) and are repeated here as controls.

In_91.1 some_91.2 cases_91.3,_91.4 similar_91.5 experiments_91.6 have_91.7 been_91.8 reported_91.9 by_91.10 Parham_91.11 et_91.12 al_91.13._91.14 (_91.152001_91.16)_91.17 (_91.18indicated_91.19 by_91.20 “_91.21a_91.22”_91.23)_91.24 and_91.25 are_91.26 repeated_91.27 here_91.28 as_91.29 controls_91.30._91.31

Aggregation:

Aggregation_92.1:_92.2

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-borne_93.1 M-GFP_93.2 fusions_93.3 of_93.4 the_93.5 indicated_93.6 Sup35p_93.7 N_93.8 domain_93.9 fragments_93.10 (_93.11green_93.12)_93.13 were_93.14 overexpressed_93.15 in_93.16 a_93.17 [_93.18psi_93.19–_93.20]_93.21 [_93.22PIN_93.23+_93.24]_93.25 strain_93.26 and_93.27 examined_93.28 for_93.29 fluorescent_93.30 focus_93.31 formation_93.32._93.33

The symbol “+” indicates that 10% or more of cells displayed aggregates.

The_94.1 symbol_94.2 “_94.3+_94.4”_94.5 indicates_94.6 that_94.7 10% or_94.8 more_94.9 of_94.10 cells_94.11 displayed_94.12 aggregates_94.13._94.14

Sup351–57-M-GFP displayed a lower frequency of aggregation (approximately 1%).

Sup351_95.1–_95.257-M-GFP_95.3 displayed_95.4 a_95.5 lower_95.6 frequency_95.7 of_95.8 aggregation_95.9 (_95.10approximately_95.11 1%)_95.12._95.13

Induction:

Induction_96.1:_96.2

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+].

Strains_97.1 from_97.2 the_97.3 aggregation_97.4 experiment_97.5 were_97.6 plated_97.7 onto_97.8 SD-ade_97.9 medium_97.10 and_97.11 scored_97.12 for_97.13 growth_97.14 to_97.15 test_97.16 whether_97.17 aggregates_97.18 of_97.19 truncated_97.20 protein_97.21 (_97.22green_97.23)_97.24 convert_97.25 chromosomally_97.26 encoded_97.27 protein_97.28 (_97.29blue_97.30)_97.31 to_97.32 [_97.33PSI_97.34+_97.35]_97.36._97.37

The symbol “+” indicates approximately 5–10% conversion frequency.

The_98.1 symbol_98.2 “_98.3+_98.4”_98.5 indicates_98.6 approximately_98.7 5_98.8–_98.910% conversion_98.10 frequency_98.11._98.12

Consistent with the aggregation experiment, Sup351–57-M-GFP displayed a lower frequency of [PSI+] induction (approximately 1%).

Consistent_99.1 with_99.2 the_99.3 aggregation_99.4 experiment_99.5,_99.6 Sup351_99.7–_99.857-M-GFP_99.9 displayed_99.10 a_99.11 lower_99.12 frequency_99.13 of_99.14 [_99.15PSI_99.16+_99.17]_99.18 induction_99.19 (_99.20approximately_99.21 1%)_99.22._99.23

Decoration:

Decoration_100.1:_100.2

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).

Indicated_101.1 proteins_101.2 were_101.3 expressed_101.4 as_101.5 –_101.6M-GFP_101.7 fusions_101.8 in_101.9 [_101.10PSI_101.11+_101.12]_101.13 [_101.14PIN_101.15+_101.16]_101.17 cells_101.18,_101.19 which_101.20 were_101.21 examined_101.22 to_101.23 determine_101.24 whether_101.25 GFP-labeled_101.26 truncations_101.27 (_101.28green_101.29)_101.30 decorate_101.31 preexisting_101.32 aggregates_101.33 of_101.34 full-length_101.35 Sup35p_101.36 (_101.37blue_101.38)_101.39._101.40

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 “*”).

Curiously_102.1,_102.2 Sup351_102.3–_102.449-M-GFP_102.5 in_102.6 [_102.7PSI_102.8+_102.9]_102.10 cells_102.11 formed_102.12 abnormally_102.13 large_102.14 “_102.15ribbon_102.16”_102.17 aggregates_102.18 of_102.19 the_102.20 kind_102.21 typically_102.22 observed_102.23 during_102.24 de_102.25 novo_102.26 [_102.27PSI_102.28+_102.29]_102.30 induction_102.31;_102.32 furthermore_102.33,_102.34 approximately_102.35 10% of_102.36 the_102.37 cells_102.38 reverted_102.39 to_102.40 [_102.41psi_102.42–_102.43]_102.44 (_102.45indicated_102.46 by_102.47 “_102.48*_102.49”_102.50)_102.51._102.52

Thus, this truncation was a potent dominant PNM mutant.

Thus_103.1,_103.2 this_103.3 truncation_103.4 was_103.5 a_103.6 potent_103.7 dominant_103.8 PNM_103.9 mutant_103.10._103.11

Maintenance:

Maintenance_104.1:_104.2

A SUP35-deleted [PSI+] [PIN+] bearing wild-type SUP35 maintainer (blue) was transformed with maintainer plasmids containing the indicated truncation (purple).

A_105.1 SUP35-deleted_105.2 [_105.3PSI_105.4+_105.5]_105.6 [_105.7PIN_105.8+_105.9]_105.10 bearing_105.11 wild-type_105.12 SUP35_105.13 maintainer_105.14 (_105.15blue_105.16)_105.17 was_105.18 transformed_105.19 with_105.20 maintainer_105.21 plasmids_105.22 containing_105.23 the_105.24 indicated_105.25 truncation_105.26 (_105.27purple_105.28)_105.29._105.30

The wild-type maintainer was lost by counterselection, and the resulting strain was tested for [PSI+] by color and growth on SD-ade medium.

The_106.1 wild-type_106.2 maintainer_106.3 was_106.4 lost_106.5 by_106.6 counterselection_106.7,_106.8 and_106.9 the_106.10 resulting_106.11 strain_106.12 was_106.13 tested_106.14 for_106.15 [_106.16PSI_106.17+_106.18]_106.19 by_106.20 color_106.21 and_106.22 growth_106.23 on_106.24 SD-ade_106.25 medium_106.26._106.27

The Sup351–93 mutant displayed an intermediate pink color and grew poorly on SD-ade medium, as previously reported (Parham et al. 2001).

The_107.1 Sup351_107.2–_107.393_107.4 mutant_107.5 displayed_107.6 an_107.7 intermediate_107.8 pink_107.9 color_107.10 and_107.11 grew_107.12 poorly_107.13 on_107.14 SD-ade_107.15 medium_107.16,_107.17 as_107.18 previously_107.19 reported_107.20 (_107.21Parham_107.22 et_107.23 al_107.24._107.25 2001_107.26)_107.27._107.28

Note:

Note_108.1:_108.2

King (2001) reports that Sup351–61-GFP fusion could decorate [PSI+] aggregates in certain strains and could induce [PSI+] de novo when overexpressed.

King_109.1 (_109.22001_109.3)_109.4 reports_109.5 that_109.6 Sup351_109.7–_109.861-GFP_109.9 fusion_109.10 could_109.11 decorate_109.12 [_109.13PSI_109.14+_109.15]_109.16 aggregates_109.17 in_109.18 certain_109.19 strains_109.20 and_109.21 could_109.22 induce_109.23 [_109.24PSI_109.25+_109.26]_109.27 de_109.28 novo_109.29 when_109.30 overexpressed_109.31._109.32

Instead, the PNM2-1 strain shows a marked defect in the inheritance of [PSI+].

Instead_110.1,_110.2 the_110.3 PNM2-1_110.4 strain_110.5 shows_110.6 a_110.7 marked_110.8 defect_110.9 in_110.10 the_110.11 inheritance_110.12 of_110.13 [_110.14PSI_110.15+_110.16]_110.17._110.18

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).

When_111.1 the_111.2 wild-type_111.3 SUP35_111.4 gene_111.5 of_111.6 a_111.7 [_111.8PSI_111.9+_111.10]_111.11 strain_111.12 was_111.13 replaced_111.14 with_111.15 PNM2-1_111.16,_111.17 the_111.18 strain_111.19 retained_111.20 the_111.21 prion_111.22 on_111.23 synthetic_111.24 defined_111.25 (_111.26SD_111.27)_111.28 yeast_111.29 medium_111.30 that_111.31 selected_111.32 for_111.33 [_111.34PSI_111.35+_111.36]_111.37 (_111.38SD-ade_111.39 medium_111.40)_111.41 but_111.42 reverted_111.43 to_111.44 [_111.45psi_111.46–_111.47]_111.48 at_111.49 a_111.50 high_111.51 frequency_111.52 in_111.53 nonselective_111.54 YEPD_111.55 medium_111.56,_111.57 resulting_111.58 in_111.59 sectored_111.60 colonies_111.61 (_111.62Figure_111.63 4D_111.64)_111.65._111.66

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).

We_112.1 measured_112.2 the_112.3 rate_112.4 of_112.5 [_112.6PSI_112.7+_112.8]_112.9 loss_112.10 in_112.11 a_112.12 PNM2-1_112.13 strain_112.14 by_112.15 growing_112.16 it_112.17 in_112.18 YEPD_112.19 medium_112.20 and_112.21,_112.22 at_112.23 various_112.24 time_112.25 points_112.26,_112.27 plating_112.28 aliquots_112.29 of_112.30 the_112.31 culture_112.32 onto_112.33 SD-ade_112.34 medium_112.35 to_112.36 determine_112.37 the_112.38 fraction_112.39 of_112.40 cells_112.41 that_112.42 had_112.43 retained_112.44 [_112.45PSI_112.46+_112.47]_112.48 (_112.49Figure_112.50 4E_112.51)_112.52._112.53

A wild-type strain retained [PSI+] in all of the cells throughout the experiment.

A_113.1 wild-type_113.2 strain_113.3 retained_113.4 [_113.5PSI_113.6+_113.7]_113.8 in_113.9 all_113.10 of_113.11 the_113.12 cells_113.13 throughout_113.14 the_113.15 experiment_113.16._113.17

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.

By_114.1 contrast_114.2,_114.3 in_114.4 the_114.5 PNM2-1_114.6 strain_114.7 the_114.8 fraction_114.9 of_114.10 [_114.11PSI_114.12+_114.13]_114.14 cells_114.15 decreased_114.16 rapidly_114.17 while_114.18 the_114.19 cells_114.20 grew_114.21 logarithmically_114.22,_114.23 but_114.24 remained_114.25 at_114.26 a_114.27 constant_114.28 level_114.29 when_114.30 the_114.31 cells_114.32 entered_114.33 stationary_114.34 phase_114.35._114.36

These findings indicate that PNM2-1 acts to eliminate [PSI+] in dividing cells, consistent with a defect in prion replication.

These_115.1 findings_115.2 indicate_115.3 that_115.4 PNM2-1_115.5 acts_115.6 to_115.7 eliminate_115.8 [_115.9PSI_115.10+_115.11]_115.12 in_115.13 dividing_115.14 cells_115.15,_115.16 consistent_115.17 with_115.18 a_115.19 defect_115.20 in_115.21 prion_115.22 replication_115.23._115.24

We next used a recently described assay to measure the number of heritable prion seeds (propagons) in a PNM2-1 strain.

We_116.1 next_116.2 used_116.3 a_116.4 recently_116.5 described_116.6 assay_116.7 to_116.8 measure_116.9 the_116.10 number_116.11 of_116.12 heritable_116.13 prion_116.14 seeds_116.15 (_116.16propagons_116.17)_116.18 in_116.19 a_116.20 PNM2-1_116.21 strain_116.22._116.23

Here, prion replication is inhibited by GuHCl treatment.

Here_117.1,_117.2 prion_117.3 replication_117.4 is_117.5 inhibited_117.6 by_117.7 GuHCl_117.8 treatment_117.9._117.10

As the cells divide, preexisting propagons are diluted but not destroyed.

As_118.1 the_118.2 cells_118.3 divide_118.4,_118.5 preexisting_118.6 propagons_118.7 are_118.8 diluted_118.9 but_118.10 not_118.11 destroyed_118.12._118.13

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).

The_119.1 number_119.2 of_119.3 propagons_119.4 present_119.5 in_119.6 a_119.7 colony_119.8 arising_119.9 from_119.10 a_119.11 single_119.12 cell_119.13 is_119.14 then_119.15 evaluated_119.16 by_119.17 removing_119.18 the_119.19 GuHCl_119.20 prion_119.21 replication_119.22 block_119.23 after_119.24 a_119.25 large_119.26 number_119.27 (_119.2810_119.29 or_119.30 more_119.31)_119.32 of_119.33 cell_119.34 divisions_119.35 and_119.36 counting_119.37 the_119.38 total_119.39 number_119.40 of_119.41 [_119.42PSI_119.43+_119.44]_119.45 cells_119.46 in_119.47 that_119.48 colony_119.49 (_119.50Cox_119.51 et_119.52 al_119.53._119.54 2003_119.55)_119.56._119.57

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).

Whereas_120.1 a_120.2 wild-type_120.3 strain_120.4 had_120.5 a_120.6 median_120.7 of_120.8 92_120.9 (_120.10n_120.11 = 24_120.12)_120.13 propagons_120.14 per_120.15 cell_120.16,_120.17 the_120.18 PNM2-1_120.19 strain_120.20 had_120.21 dramatically_120.22 fewer_120.23:_120.24 41_120.25 of_120.26 50_120.27 cells_120.28 had_120.29 no_120.30 [_120.31PSI_120.32+_120.33]_120.34 propagons_120.35 at_120.36 all_120.37 (_120.38i_120.39._120.40e_120.41._120.42,_120.43 were_120.44 [_120.45psi_120.46–_120.47]_120.48)_120.49,_120.50 and_120.51 among_120.52 the_120.53 remaining_120.54 nine_120.55 [_120.56PSI_120.57+_120.58]_120.59 cells_120.60,_120.61 the_120.62 median_120.63 propagon_120.64 number_120.65 was_120.66 six_120.67 (_120.68Figure_120.69 4F_120.70)_120.71._120.72

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.

Thus_121.1,_121.2 although_121.3 a_121.4 PNM2-1_121.5 strain_121.6 can_121.7 harbor_121.8 [_121.9PSI_121.10+_121.11]_121.12 prions_121.13,_121.14 a_121.15 defect_121.16 in_121.17 propagon_121.18 replication_121.19 causes_121.20 mitotic_121.21 instability_121.22,_121.23 demonstrating_121.24 the_121.25 importance_121.26 of_121.27 oligopeptide_121.28 repeat_121.29 2_121.30 in_121.31 prion_121.32 replication_121.33 or_121.34 segregation_121.35._121.36

Figure 4.

Figure_122.1 4_122.2._122.3

PNM2–1 (G58D) Prevents Inheritance But Not Aggregation of Sup35p Prions

PNM2_123.1–_123.21_123.3 (_123.4G58D_123.5)_123.6 Prevents_123.7 Inheritance_123.8 But_123.9 Not_123.10 Aggregation_123.11 of_123.12 Sup35p_123.13 Prions_123.14

(A) PNM2-1 protein can seed [PSI+].

(_124.1A_124.2)_124.3 PNM2-1_124.4 protein_124.5 can_124.6 seed_124.7 [_124.8PSI_124.9+_124.10]_124.11._124.12

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.

A_125.1 Sup35p_125.2 inducer_125.3 containing_125.4 the_125.5 PNM2-1_125.6 (_125.7G58D_125.8)_125.9 mutation_125.10 was_125.11 overexpressed_125.12 in_125.13 [_125.14psi_125.15–_125.16]_125.17 [_125.18PIN_125.19+_125.20]_125.21 cells_125.22;_125.23 shown_125.24 are_125.25 cells_125.26 (_125.27inset_125.28)_125.29 with_125.30 representative_125.31 fluorescent_125.32 foci_125.33,_125.34 which_125.35 were_125.36 the_125.37 same_125.38 in_125.39 frequency_125.40 and_125.41 appearance_125.42 as_125.43 cells_125.44 with_125.45 a_125.46 wild-type_125.47 inducer_125.48._125.49

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+.

Cells_126.1 overexpressing_126.2 inducer_126.3 versions_126.4 of_126.5 wild-type_126.6 Sup35p_126.7 (_126.8SUP_126.9)_126.10,_126.11 an_126.12 aggregation-defective_126.13 N-terminal_126.14 truncation_126.15 (_126.16Δ1_126.17–_126.1838_126.19)_126.20,_126.21 and_126.22 PNM2-1_126.23 were_126.24 plated_126.25 and_126.26 scored_126.27 for_126.28 Ade_126.29+_126.30._126.31

Approximately 1000 colonies were counted.

Approximately_127.1 1000_127.2 colonies_127.3 were_127.4 counted_127.5._127.6

(B) PNM2-1 protein polymerization is similar to that of wild-type protein.

(_128.1B_128.2)_128.3 PNM2-1_128.4 protein_128.5 polymerization_128.6 is_128.7 similar_128.8 to_128.9 that_128.10 of_128.11 wild-type_128.12 protein_128.13._128.14

(C) Preformed PNM2-1 polymers seed wild-type and PNM2-1 monomers with comparable efficiency.

(_129.1C_129.2)_129.3 Preformed_129.4 PNM2-1_129.5 polymers_129.6 seed_129.7 wild-type_129.8 and_129.9 PNM2-1_129.10 monomers_129.11 with_129.12 comparable_129.13 efficiency_129.14._129.15

Endpoint PNM2-1 polymers were used to seed fresh reactions.

Endpoint_130.1 PNM2-1_130.2 polymers_130.3 were_130.4 used_130.5 to_130.6 seed_130.7 fresh_130.8 reactions_130.9._130.10

(D) PNM2-1 displays a partially dominant, incompletely penetrant defect in [PSI+] maintenance.

(_131.1D_131.2)_131.3 PNM2-1_131.4 displays_131.5 a_131.6 partially_131.7 dominant_131.8,_131.9 incompletely_131.10 penetrant_131.11 defect_131.12 in_131.13 [_131.14PSI_131.15+_131.16]_131.17 maintenance_131.18._131.19

[psi–] (1) and [PSI+] (2) SUP35::TRP1 pSUP35 controls are shown.

[_132.1psi_132.2–_132.3]_132.4 (_132.51_132.6)_132.7 and_132.8 [_132.9PSI_132.10+_132.11]_132.12 (_132.132_132.14)_132.15 SUP35_132.16:_132.17:_132.18TRP1_132.19 pSUP35_132.20 controls_132.21 are_132.22 shown_132.23._132.24

[PSI+] [PIN+] SUP35::TRP1 pSUP35 was transformed with a second maintainer expressing PNM2-1 (3).

[_133.1PSI_133.2+_133.3]_133.4 [_133.5PIN_133.6+_133.7]_133.8 SUP35_133.9:_133.10:_133.11TRP1_133.12 pSUP35_133.13 was_133.14 transformed_133.15 with_133.16 a_133.17 second_133.18 maintainer_133.19 expressing_133.20 PNM2-1_133.21 (_133.223_133.23)_133.24._133.25

The wild-type maintainer (pSUP35) was then lost through counterselection (4).

The_134.1 wild-type_134.2 maintainer_134.3 (_134.4pSUP35_134.5)_134.6 was_134.7 then_134.8 lost_134.9 through_134.10 counterselection_134.11 (_134.124_134.13)_134.14._134.15

Red sectors from (4) were isolated, retransformed with the wild-type maintainer, and allowed to lose the PNM2-1 maintainer (5).

Red_135.1 sectors_135.2 from_135.3 (_135.44_135.5)_135.6 were_135.7 isolated_135.8,_135.9 retransformed_135.10 with_135.11 the_135.12 wild-type_135.13 maintainer_135.14,_135.15 and_135.16 allowed_135.17 to_135.18 lose_135.19 the_135.20 PNM2-1_135.21 maintainer_135.22 (_135.235_135.24)_135.25._135.26

(E) Mitotic instability of [PSI+] in the PNM2-1 strain.

(_136.1E_136.2)_136.3 Mitotic_136.4 instability_136.5 of_136.6 [_136.7PSI_136.8+_136.9]_136.10 in_136.11 the_136.12 PNM2-1_136.13 strain_136.14._136.15

A pink (Ade+) [PSI+] [PIN+] PNM2-1 isolate was grown to log phase in SD-ade liquid then shifted into nonselective (YEPD) medium.

A_137.1 pink_137.2 (_137.3Ade_137.4+_137.5)_137.6 [_137.7PSI_137.8+_137.9]_137.10 [_137.11PIN_137.12+_137.13]_137.14 PNM2-1_137.15 isolate_137.16 was_137.17 grown_137.18 to_137.19 log_137.20 phase_137.21 in_137.22 SD-ade_137.23 liquid_137.24 then_137.25 shifted_137.26 into_137.27 nonselective_137.28 (_137.29YEPD_137.30)_137.31 medium_137.32._137.33

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).

At_138.1 indicated_138.2 time_138.3 points_138.4,_138.5 aliquots_138.6 were_138.7 plated_138.8 onto_138.9 SD-ade_138.10 and_138.11 YEPD_138.12 media_138.13 to_138.14 determine_138.15 the_138.16 fraction_138.17 of_138.18 [_138.19PSI_138.20+_138.21]_138.22 cells_138.23 (_138.24minimum_138.25 of_138.26 200_138.27 colonies_138.28 counted_138.29 per_138.30 time_138.31 point_138.32)_138.33._138.34

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%).

Whereas_139.1 a_139.2 wild-type_139.3 control_139.4 remained_139.5 [_139.6PSI_139.7+_139.8]_139.9 through_139.10 the_139.11 experiment_139.12,_139.13 the_139.14 PNM2-1_139.15 strain_139.16 rapidly_139.17 lost_139.18 [_139.19PSI_139.20+_139.21]_139.22 during_139.23 logarithmic_139.24 growth_139.25;_139.26 during_139.27 stationary_139.28 phase_139.29 (_139.3018_139.31 h_139.32 and_139.33 beyond_139.34)_139.35,_139.36 the_139.37 percentage_139.38 of_139.39 [_139.40PSI_139.41+_139.42]_139.43 PNM2-1_139.44 strains_139.45 remained_139.46 unchanged_139.47 (_139.48approximately_139.49 5%)_139.50._139.51

(F) Propagon count of PNM2-1 vs. wild-type [PSI+] strains.

(_140.1F_140.2)_140.3 Propagon_140.4 count_140.5 of_140.6 PNM2-1_140.7 vs_140.8._140.9 wild-type_140.10 [_140.11PSI_140.12+_140.13]_140.14 strains_140.15._140.16

The majority of PNM2-1 cells had no [PSI+] propagons (i.e., were [psi–]).

The_141.1 majority_141.2 of_141.3 PNM2-1_141.4 cells_141.5 had_141.6 no_141.7 [_141.8PSI_141.9+_141.10]_141.11 propagons_141.12 (_141.13i_141.14._141.15e_141.16._141.17,_141.18 were_141.19 [_141.20psi_141.21–_141.22]_141.23)_141.24._141.25

In both strains, a small number of “jackpot” cells contained over 200 propagons; see Cox et al. (2003).

In_142.1 both_142.2 strains_142.3,_142.4 a_142.5 small_142.6 number_142.7 of_142.8 “_142.9jackpot_142.10”_142.11 cells_142.12 contained_142.13 over_142.14 200_142.15 propagons_142.16;_142.17 see_142.18 Cox_142.19 et_142.20 al_142.21._142.22 (_142.232003_142.24)_142.25._142.26

Design of Novel Prion Domains

Design_143.1 of_143.2 Novel_143.3 Prion_143.4 Domains_143.5

Our data suggested that the formation and inheritance of prions involve distinct regions of Sup35p and New1p prion domains.

Our_144.1 data_144.2 suggested_144.3 that_144.4 the_144.5 formation_144.6 and_144.7 inheritance_144.8 of_144.9 prions_144.10 involve_144.11 distinct_144.12 regions_144.13 of_144.14 Sup35p_144.15 and_144.16 New1p_144.17 prion_144.18 domains_144.19._144.20

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).

To_145.1 assess_145.2 the_145.3 interchangeability_145.4 of_145.5 these_145.6 prion_145.7 domain_145.8 components_145.9,_145.10 we_145.11 constructed_145.12 a_145.13 chimeric_145.14 prion_145.15 domain_145.16,_145.17 termed_145.18 F_145.19,_145.20 in_145.21 which_145.22 the_145.23 aggregation-determining_145.24 NYN_145.25 repeat_145.26 of_145.27 New1p_145.28 was_145.29 fused_145.30 to_145.31 the_145.32 oligopeptide_145.33 repeats_145.34 of_145.35 Sup35p_145.36 (_145.37Figure_145.38 5A_145.39)_145.40._145.41

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.

While_146.1 initially_146.2 soluble_146.3 and_146.4 active_146.5,_146.6 a_146.7 fusion_146.8 of_146.9 F_146.10 and_146.11 the_146.12 Sup35p_146.13 M_146.14 and_146.15 C_146.16 domains_146.17 (_146.18F-M-C_146.19)_146.20 could_146.21 be_146.22 converted_146.23 into_146.24 an_146.25 aggregated_146.26 state_146.27,_146.28 termed_146.29 [_146.30F_146.31+_146.32]_146.33,_146.34 after_146.35 transient_146.36 overexpression_146.37 of_146.38 F-M-GFP_146.39._146.40

As with [NU+], [F+] induction did not require [PIN+] (data not shown).

As_147.1 with_147.2 [_147.3NU_147.4+_147.5]_147.6,_147.7 [_147.8F_147.9+_147.10]_147.11 induction_147.12 did_147.13 not_147.14 require_147.15 [_147.16PIN_147.17+_147.18]_147.19 (_147.20data_147.21 not_147.22 shown_147.23)_147.24._147.25

[F+] could be eliminated by GuHCl treatment (Figure 5B) and was inherited in a dominant, non-Mendelian manner (Figure 5C).

[_148.1F_148.2+_148.3]_148.4 could_148.5 be_148.6 eliminated_148.7 by_148.8 GuHCl_148.9 treatment_148.10 (_148.11Figure_148.12 5B_148.13)_148.14 and_148.15 was_148.16 inherited_148.17 in_148.18 a_148.19 dominant_148.20,_148.21 non-Mendelian_148.22 manner_148.23 (_148.24Figure_148.25 5C_148.26)_148.27._148.28

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).

As_149.1 with_149.2 Sup35p_149.3 in_149.4 a_149.5 [_149.6PSI_149.7+_149.8]_149.9 strain_149.10,_149.11 F-M-C_149.12 protein_149.13 in_149.14 [_149.15F_149.16+_149.17]_149.18 but_149.19 not_149.20 in_149.21 [_149.22f_149.23 –_149.24]_149.25 extracts_149.26 sedimented_149.27 entirely_149.28 to_149.29 the_149.30 pellet_149.31 fraction_149.32 following_149.33 high-speed_149.34 centrifugation_149.35 (_149.36Figure_149.37 5D_149.38)_149.39._149.40

Thus, [F+] results from a prion state of F-M-C.

Thus_150.1,_150.2 [_150.3F_150.4+_150.5]_150.6 results_150.7 from_150.8 a_150.9 prion_150.10 state_150.11 of_150.12 F-M-C_150.13._150.14

We next explored the specificity of [F+] prion seeding.

We_151.1 next_151.2 explored_151.3 the_151.4 specificity_151.5 of_151.6 [_151.7F_151.8+_151.9]_151.10 prion_151.11 seeding_151.12._151.13

Overexpression of the Sup35p prion domain did not induce [F+]; conversely, F-M-GFP overexpression did not induce [PSI+] (Figure 5E).

Overexpression_152.1 of_152.2 the_152.3 Sup35p_152.4 prion_152.5 domain_152.6 did_152.7 not_152.8 induce_152.9 [_152.10F_152.11+_152.12]_152.13;_152.14 conversely_152.15,_152.16 F-M-GFP_152.17 overexpression_152.18 did_152.19 not_152.20 induce_152.21 [_152.22PSI_152.23+_152.24]_152.25 (_152.26Figure_152.27 5E_152.28)_152.29._152.30

However, F-M-GFP readily induced [NU+], indicating that mismatched sequences outside of the aggregating region did not prevent cross-interactions between heterologous proteins.

However_153.1,_153.2 F-M-GFP_153.3 readily_153.4 induced_153.5 [_153.6NU_153.7+_153.8]_153.9,_153.10 indicating_153.11 that_153.12 mismatched_153.13 sequences_153.14 outside_153.15 of_153.16 the_153.17 aggregating_153.18 region_153.19 did_153.20 not_153.21 prevent_153.22 cross-interactions_153.23 between_153.24 heterologous_153.25 proteins_153.26._153.27

Interestingly, overexpression of New11–53-GFP induced Ade+ colonies in the [f –] strain, but this adenine prototrophy proved unstable.

Interestingly_154.1,_154.2 overexpression_154.3 of_154.4 New11_154.5–_154.653-GFP_154.7 induced_154.8 Ade_154.9+_154.10 colonies_154.11 in_154.12 the_154.13 [_154.14f_154.15 –_154.16]_154.17 strain_154.18,_154.19 but_154.20 this_154.21 adenine_154.22 prototrophy_154.23 proved_154.24 unstable_154.25._154.26

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).

We_155.1 also_155.2 examined_155.3 the_155.4 ability_155.5 of_155.6 preexisting_155.7 prion_155.8 aggregates_155.9 to_155.10 recruit_155.11 different_155.12 prion-forming_155.13 proteins_155.14 using_155.15 an_155.16 antisuppression_155.17 assay_155.18 (_155.19Santoso_155.20 et_155.21 al_155.22._155.23 2000_155.24)_155.25 (_155.26Figure_155.27 5F_155.28)_155.29._155.30

[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).

[_156.1PSI_156.2+_156.3]_156.4,_156.5 [_156.6F_156.7+_156.8]_156.9,_156.10 and_156.11 [_156.12NU_156.13+_156.14]_156.15 strains_156.16 were_156.17 transformed_156.18 with_156.19 Sup35p_156.20–_156.21,_156.22 F-M-C_156.23–_156.24 or_156.25 New11_156.26–_156.27153-M-C_156.28–_156.29encoding_156.30 plasmids_156.31;_156.32 the_156.33 color_156.34 of_156.35 the_156.36 resulting_156.37 colonies_156.38 indicates_156.39 whether_156.40 the_156.41 second_156.42 maintainer_156.43 protein_156.44 is_156.45 soluble_156.46 (_156.47red_156.48)_156.49 or_156.50 aggregates_156.51 as_156.52 a_156.53 result_156.54 of_156.55 the_156.56 resident_156.57 prion_156.58 (_156.59pink/white_156.60)_156.61._156.62

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.

Consistent_157.1 with_157.2 the_157.3 induction_157.4 data_157.5,_157.6 F-M-C_157.7 and_157.8 New11_157.9–_157.10153-M-C_157.11 were_157.12 not_157.13 incorporated_157.14 into_157.15 [_157.16PSI_157.17+_157.18]_157.19 aggregates_157.20;_157.21 likewise_157.22,_157.23 Sup35p_157.24 did_157.25 not_157.26 interact_157.27 with_157.28 [_157.29F_157.30+_157.31]_157.32 or_157.33 [_157.34NU_157.35+_157.36]_157.37 aggregates_157.38._157.39

However, [F+] prions recruited New11–153-M-C and, to a lesser extent, [NU+] recruited F-M-C.

However_158.1,_158.2 [_158.3F_158.4+_158.5]_158.6 prions_158.7 recruited_158.8 New11_158.9–_158.10153-M-C_158.11 and_158.12,_158.13 to_158.14 a_158.15 lesser_158.16 extent_158.17,_158.18 [_158.19NU_158.20+_158.21]_158.22 recruited_158.23 F-M-C_158.24._158.25

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.

Thus_159.1,_159.2 F_159.3 and_159.4 New1p_159.5 prion_159.6 domains_159.7 can_159.8 cross-interact_159.9 during_159.10 de_159.11 novo_159.12 induction_159.13 and_159.14 at_159.15 normal_159.16 levels_159.17 of_159.18 expression_159.19,_159.20 indicating_159.21 that_159.22 the_159.23 NYN_159.24 repeat_159.25 is_159.26 sufficient_159.27 to_159.28 specify_159.29 homotypic_159.30 interaction_159.31 between_159.32 two_159.33 otherwise_159.34 distinct_159.35 prion_159.36 domains_159.37._159.38

Figure 5.

Figure_160.1 5_160.2._160.3

F, A New1p–Sup35p Chimera, Shows Prion Characteristics of New1p

F_161.1,_161.2 A_161.3 New1p_161.4–_161.5Sup35p_161.6 Chimera_161.7,_161.8 Shows_161.9 Prion_161.10 Characteristics_161.11 of_161.12 New1p_161.13

(A) Schematic diagram illustrating the construction of chimera F.

(_162.1A_162.2)_162.3 Schematic_162.4 diagram_162.5 illustrating_162.6 the_162.7 construction_162.8 of_162.9 chimera_162.10 F_162.11._162.12

(B) Chimera F forms a prion, [F+].

(_163.1B_163.2)_163.3 Chimera_163.4 F_163.5 forms_163.6 a_163.7 prion_163.8,_163.9 [_163.10F_163.11+_163.12]_163.13._163.14

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.

The_164.1 SUP35_164.2 gene_164.3 in_164.4 a_164.5 [_164.6psi_164.7–_164.8]_164.9 [_164.10pin_164.11–_164.12]_164.13 strain_164.14 was_164.15 replaced_164.16 with_164.17 the_164.18 F-M-C_164.19 fusion_164.20;_164.21 after_164.22 transient_164.23 overexpression_164.24 of_164.25 F-M-GFP_164.26,_164.27 approximately_164.28 10% of_164.29 these_164.30 cells_164.31 converted_164.32 from_164.33 an_164.34 Ade- (_164.35[_164.36f_164.37 –_164.38]_164.39)_164.40 to_164.41 an_164.42 Ade_164.43+_164.44 (_164.45[_164.46F_164.47+_164.48]_164.49)_164.50 state_164.51._164.52

Shown are examples of[f –] and [F+] strains, before and after GuHCl treatment, along with [psi–] and [PSI+] controls.

Shown_165.1 are_165.2 examples_165.3 of_165.4[_165.5f_165.6 –_165.7]_165.8 and_165.9 [_165.10F_165.11+_165.12]_165.13 strains_165.14,_165.15 before_165.16 and_165.17 after_165.18 GuHCl_165.19 treatment_165.20,_165.21 along_165.22 with_165.23 [_165.24psi_165.25–_165.26]_165.27 and_165.28 [_165.29PSI_165.30+_165.31]_165.32 controls_165.33._165.34

(C) Non-Mendelian inheritance of [F+].

(_166.1C_166.2)_166.3 Non-Mendelian_166.4 inheritance_166.5 of_166.6 [_166.7F_166.8+_166.9]_166.10._166.11

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.

A_167.1 diploid_167.2 made_167.3 by_167.4 mating_167.5 a_167.6 [_167.7F_167.8+_167.9]_167.10 MATα_167.11 strain_167.12 against_167.13 an_167.14 [_167.15f_167.16 –_167.17]_167.18 MATα_167.19 displayed