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J. Biol. Chem., Vol. 282, Issue 5, 3004-3013, February 2, 2007
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12
13
From the
School of Biology and Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0230 and the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
Received for publication, October 11, 2006 , and in revised form, November 21, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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on [PSI+] loss was increased by depletion of the Hsp70 chaperone Ssb but was not influenced by depletion of Ubp6. This indicates that Ubc4 affects [PSI+] loss via a pathway that is the same as the one affected by Ubp6 but not by Ssb. In the presence of Rnq1 protein, ubc4 also facilitates spontaneous de novo formation of [PSI+]. This stimulation is independent of [PIN+], the prion isoform of Rnq1. Numerous attempts failed to detect ubiquitinated Sup35 in the yeast extracts. While ubc4 and other alterations of ubiquitin system used in this work cause slight induction of some Hsps, these changes are insufficient to explain their effect on [PSI+]. However, ubc4 increases the proportion of the Hsp70 chaperone Ssa bound to Sup35, suggesting that misfolded Sup35 is either more abundant or more accessible to the chaperones in the absence of Ubc4. The proportion of [PSI+] cells containing large aggregated Sup35 structures is also increased by ubc4. We propose that UPS alterations induce an adaptive response, resulting in accumulation of the large "aggresome"-like aggregates that promote de novo prion generation and prion recovery from the chaperone treatment. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Degradation of abnormal or damaged proteins occurs, at least in part, via the ubiquitin-proteasome system (UPS)4 (4). Targeting of a protein for degradation to the proteasome requires its conjugation to a 76-amino acid protein named ubiquitin (Ub) through the sequential action of several enzymes, including the Ub-conjugating enzymes (Ubc). This process can be reversed by the action of various deubiquitinating enzymes, including Ub-specific processing proteases (Ubp) that disassemble Ub conjugates and release free Ub (5). Mutations in different UPS components have been found to be associated with Alzheimer and Parkinson diseases (6). UPS failure can lead to aggregation of impaired proteins; in turn, aggregate formation is known to inhibit UPS function (7). Although proteasome inhibitors affect the turnover of mammalian prion proteins, and Ub is found in intracellular deposits of prion aggregates (8–10), the role of Ub-mediated proteolysis in prion formation is not defined.
Several yeast proteins possess prion properties (11–13). All known yeast prions contain QN-rich stretches, somewhat resembling poly(Q) stretches involved in aggregation disorders such as Huntington disease. Unlike the mammalian prion protein PrP, yeast prions do not kill cells, although some of them appear to be pathogenic in a longer run (14–16). Prion formation can antagonize the normal cellular function of a yeast protein, thus producing changes in phenotype that mimic conventional loss-of-function mutations. The yeast non-Mendelian element [PSI+] is a prion isoform of the yeast translation termination factor Sup35. [PSI+] cells are partially defective in termination of translation. [PIN+], a prion isoform of another protein of yet unknown function, Rnq1 (17), is usually needed for the de novo formation of [PSI+] but not for propagation of pre-existing [PSI+] (18–21).
The propagation of yeast prions is modulated by chaperone proteins of the Hsp100 and Hsp70 families (11, 12). The chaperone protein Hsp104, an ATPase known to promote solubilization of aggregated heat-damaged proteins in cooperation with other chaperones (22), is required for maintenance of all known yeast prions, possibly due to its ability to break prion aggregates into the oligomeric seeds, initiating new rounds of prion propagation. Transient overproduction of Hsp104 also cures yeast cells of [PSI+], although not of the other prions. Ssa proteins of the Hsp70 family also play an important role in prion propagation. Ssa overproduction antagonizes [PSI+] curing by excess Hsp104 and promotes de novo [PSI+] formation in the [PIN+] strains (23, 24), while Ssa mutations destabilize [PSI+] (25). Another chaperone of the Hsp70 family, Ssb, consistently manifests itself as a [PSI+] antagonist (26). Hsp40 chaperones that act as co-chaperones of Hsp70s were shown to control propagation of [PIN+] (27) and modulate aggregation of heterologous poly(Q) proteins in yeast (28, 29). Mechanisms of the chaperone effects on prions are not yet completely understood. As some of the chaperones of Hsp70 and Hsp40 families were implicated in targeting misfolded proteins for Ubdependent degradation (30, 31), it is possible that some of the effects of chaperones on prions could be mediated by UPS.
We employed [PSI+] as a model to analyze the possible involvement of the UPS in modulation of prion formation, propagation, and clearance. Previously, we have shown that depletion of cellular Ub levels (by deleting the deubiquitinating enzyme Ubp6) decreases both the phenotypic manifestation of [PSI+] and de novo induction of [PSI+] by overproduced Sup35 protein in the [PIN+] background (32). Here, we demonstrate that both ubp6 and deletion of a gene coding for the major yeast Ub-conjugating enzyme, Ubc4, antagonize [PSI+] curing by excess Hsp104. Moreover, ubc4 also significantly increased spontaneous formation of [PSI+]. Our data implicate the UPS as one of the major modulators of prion formation and clearance in the yeast cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Media and Growth Conditions—Standard yeast media, cultivation conditions, procedures for yeast growth, transformation, sporulation, and tetrad analysis were used (43). Yeast cultures were grown at 30 °C unless otherwise specified. Gal medium contained 2% galactose instead of glucose. Gal+Raf medium contained 2% galactose and 2% raffinose instead of glucose. To induce expression of genes under the PCUP1 promoter, CuSO4 (50 or 100 µM) was added to the media. For drug sensitivity assay, strains were grown on YPD medium to an A600 of 1.0, and 2.5 µl of serial 10-fold dilutions were plated on YPD (1% yeast extract, 2% peptone, 2% glucose) medium with anisomycin (20 µg/ml). Growth on the plates was recorded after 2 and 5 days. The presence of [PSI+] was monitored by its ability to suppress the reporter allele ade1–14 (UGA) as described previously (33). In the absence of [PSI+], ade1–14 cultures develop red color on YPD and are unable to grow on -Ade medium. In the presence of [PSI+], these cultures develop light pink or white color on YPD and are able to grow on -Ade medium.
Assays for [PSI+] Curing—Assays for [PSI+] curing by GuHCl were performed as described previously (33). Briefly, to measure [PSI+] curing by overproduced Hsp104, yeast cultures were transformed with the Hsp104 expression vectors or empty control vectors and analyzed as outlined below. For plate assays with the plasmids expressing Hsp104 from the endogenous (PHSP104) or constitutively active (PGPD) promoters, plasmid-containing cultures were grown on the medium selective for the plasmid and then velveteen replica plated onto the medium not selective for the plasmid and lacking adenine or onto YPD medium. For plate assays with the galactose-inducible (PGAL) promoter, cultures were grown on selective medium containing Gal and then velveteen replica-plated onto selective media containing glucose but lacking adenine or onto YPD medium. For all plasmids, the efficiency of [PSI+] curing was assessed by decreased growth on -Ade medium and increased intensity of red color on YPD medium compared with the control cultures. For quantitative assays, yeast cultures containing the plasmids bearing HSP104 under PGAL promoter were pregrown in selective medium containing glucose and then inoculated into the Gal+Raf medium of the same composition and grown with shaking (200–250 rpm) at 30 °C. Aliquots were taken after specified periods of time and plated onto selective medium containing glucose. The colonies grown after 4–5 days were velveteen replica-plated onto YPD and -Ade medium. [PSI+] retention or loss was scored by color on YPD and growth on -Ade.
Assays for [PSI+] Formation—Plate assays and quantitative assays for spontaneous [PSI+] formation were performed as described previously (26). Rates of spontaneous [PSI+] formation and confidence limits were calculated by using the formulas used previously for measuring the rates of spontaneous mutations (44).
Protein Analysis and Antibodies—For measuring the levels of Sup35, chaperone proteins, and Ub, yeast cells were lysed by vortexing with glass beads (Sup35 and chaperones) or by boiling in SDS loading buffer (Ub) according to protocols described previously (24, 32, 41). Co-imunoprecipitation assays for detection of Sup35 and Ssa interaction were performed using immobilized protein A (Invitrogen) according to the published protocol (23). A ubiquitinated protein enrichment kit (Calbiochem EMD Biosciences, Inc., San Diego, CA) was used to search for ubiquitinated Sup35 according to the manufacturer protocol. Total protein extracts and immunoprecipitates were examined by Western analysis using specific antibodies. Antibodies to Sup35NM were obtained as described previously (41), and antibodies to Sup35C were the generous gift of D. Bedwell. Antibodies to Hsp104 were kindly provided by S. Lindquist. Antibodies to total Ssa, Ssa3/4, and Ssb were generously provided by E. A. Craig. Antibodies to Ydj1 and Sis1 were kindly provided by D. Cyr. We used anti-Ub antibody P4D1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), HA-specific antibody 12CA5 from Maine Biotechnology Services, anti-Pgk antibody (Molecular Probes, Inc., Eugene, OR), and anti-c-Myc antibody (Ab-1) from Calbiochem EMD Biosciences, Inc. In all experiments appropriate secondary antibodies from GE Healthcare Ltd. (Buckinhamshire, UK) were used. Western blots were developed according to the ECL detection system protocol from Pierce. Densitometry was performed on HyBlotCL autoradiography films (Denville Scientific, Inc., Metuchen, NJ) using the LabWorks 4.0 program on a UVP BioImaging system (UVP Inc., Upland, CA).
Detection of Sup35NM-GFP Aggregation in the Yeast Cells— GFP detection in live yeast cells was performed as described previously (33, 45). Yeast cultures containing the plasmid pmCUPNMsGFP were pregrown in selective medium lacking uracil for a period of 2 days and then inoculated into the same medium with 50 or 100 µM CuSO4, to induce the PCUP1 promoter (the starting concentration of cells was 2–3 x 106 cells/ml). After the specified periods of time, aliquots were taken, and proportions of transformed cells containing detectable aggregated structures were determined by using the microscope BX41 (Olympus) with the Endow GFP Bandpass Emission filter. Cultures containing the pmCUPsGFP construct that was used as a control never produced cytologically detectable aggregates in these conditions (data not shown), confirming that aggregation is specific to the Sup35-GFP fused protein.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Deletion strains and wild-type control strain were transformed with the plasmids producing Hsp104 from either the galactose-inducible (PGAL) or a strong constitutive (PGPD) promoter, as well as by empty control vectors. In the case of ubc4, we also moderately overexpressed HSP104 by providing a single plasmid-borne copy under the control of its endogenous promoter. Levels of overexpressed Hsp104 were similar in wild-type and UPS mutants as confirmed by Western blot analysis (Fig. 1A and data not shown). The efficiency of [PSI+] curing in the presence of either PGAL-HSP104 (Fig. 1A) or PGPD-HSP104 (data not shown) constructs was detected by color on YPD medium and growth on -Ade medium. Efficiency of curing was decreased in ubc4 and ubp6 single deletion strains, compared with the wild-type strain. [PSI+] loss in the presence of an extra copy of PHSP104-HSP104 was also decreased in ubc4 (data not shown). Quantitative assays employing the PGAL-HSP104 construct demonstrated that the effect of double ubc4 ubp6 deletion on [PSI+] curing by excess Hsp104 is the same as the effect of single ubc4 deletion (Fig. 1B). This result suggests that both proteins influence [PSI+] curing via one and the same pathway and that Ubc4 acts before Ubp6. Such a conclusion is in agreement with the known roles of both enzymes in the Ub-proteasome degradation pathway. Interestingly, the efficiency of [PSI+] curing by excess Hsp104 was not altered in the ubc5 strain (data not shown), despite the fact that Ubc5 is known to have a role that is very similar to and partly redundant with that of Ubc4. A double ubc4 ubc5 deletion strain was not viable in the genetic background of our strains.
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. To check if ssb1/2 and deficiencies in the UPS components influence [PSI+] loss through one and the same pathway, we compared [PSI+] curing by excess of Hsp104 in several strains: the single ubc4 deletion strain, the double ssb1/2 strain, and the triple ubc4 ssb1/2 deletion strain. The triple ubc4 ssb1/2 deletion decreased the efficiency of [PSI+] curing in comparison to both single ubc4 or double ssb1/2 deletions (Fig. 1C). The additive effect of ubc4 and ssb1/2 indicates that these mutations influence [PSI+] curing by excess Hsp104 via different pathways.
Spontaneous Formation of [PSI+] Is Increased in the ubc4 Background—Next, we checked whether spontaneous [PSI+] formation is influenced by the UPS alterations. In the [psi-] strain bearing the ade1–14 reporter allele, spontaneous [PSI+] formation can be detected as the emergence of small Ade+ revertants (papillae) on a patched colony growing on -Ade medium. Papillae are usually observed only in the presence of [PIN+], the prion form of the Rnq1 protein (18–20). The [psi- pin-] strains containing non-prion form of Rnq1 rarely papillate on -Ade, and if they do, the resulting Ade+ papillae usually contain a chromosomal mutation rather than the [PSI+] prion. Surprisingly, we observed that both [psi- PIN+] (Fig. 2A) and [psi- pin-] (Fig. 2B) strains bearing the ubc4 deletion exhibited significantly increased frequency of the spontaneous Ade+ papillae (compared with the isogenic [psi- PIN+] and [psi- pin-] UBC4+ strains, respectively). In contrast, neither ubc5 nor ubp6 exhibited an increase in papillation, in either [psi- PIN+]or[psi- pin-] backgrounds. The ubc5 [psi- pin-] strain is shown as an example in Fig. 2B. One should note that the negative result for ubp6 is somewhat ambiguous, as ubp6 is known to weaken growth of the [PSI+] strains on -Ade medium (32), so that some newly arisen Ade+ derivatives might remain unnoticed in this strain.
Increased spontaneous papillation of the ubc4 strain on -Ade media was eliminated by reintroduction of the wild-type UBC4+ gene on a plasmid (Fig. 2C). When the ubc4 [psi- PIN+] strain was mated to the isogenic [psi- pin-] UBC4+ strain of the opposite mating type, and the resulting diploid was sporulated and dissected, the increased papillation on -Ade co-segregated with the ubc4 marker (HIS3) in tetrad analysis (Fig. 4D and Table 2). These data confirm that increased papillation is due to lack of UBC4 gene rather than any independent alteration in the yeast genome.
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strains were tested for the curability of Ade+ phenotype by growth in the presence of GuHCl, an agent known to eliminate yeast prions (33, 47). While the majority of colonies obtained in the [PIN+] strains were curable by GuHCl, about 77% Ade+ colonies obtained in the UBC4+ [psi- pin-] strain were incurable, indicating that they originated from mechanisms other than prion formation, such as reversion of the ade1–14 mutation or suppressor mutation. In contrast, 94% of Ade+ colonies obtained in the ubc4 [psi- pin-] strain were curable by GuHCl. All GuHCl-curable Ade+ colonies isolated from this strain were also curable by the PGPD-HSP104 plasmid, known to eliminate [PSI+] as described above. This confirms that majority of spontaneous Ade+ colonies detected in the ubc4 [psi- pin-] strain result from [PSI+] formation rather than from suppressor or reverse mutation. Quantitative assay confirmed that the differences in rates of spontaneous [PSI+] formation between the ubc4 and wild-type strains in both [psi- PIN+] (data not shown) and [psi- pin-] (Table 3) backgrounds are statistically significant.
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strain. For this purpose, 37 independently obtained Ade+ derivatives of this strain were cured of [PSI+] by overproduction of Hsp104 from the PGPD promoter. Excess Hsp104 eliminates [PSI+] but not [PIN+] (18). Resulting [psi-] derivatives were cured of the PGPD-HSP104 plasmid and tested for the presence of [PIN+]by mating them to the [psi- pin-] strain of opposite mating type bearing the multicopy SUP35 plasmid. No [PSI+] formation was detected in the resulting diploids, indicating that all original [PSI+] derivatives remained [pin-]. Therefore, increased spontaneous formation of [PSI+] in the [pin-] ubc4 strain is not due to generation of [PIN+].
To check whether Rnq1 protein in its non-prion form influences formation of the [PSI+] prion in the ubc4 background, we deleted the RNQ1 gene in both wild-type and ubc4 [psi-] strains and found that rnq1 dramatically reduced frequency of the spontaneous [PSI+] papillae in the ubc4 strain, making it indistinguishable from the wild-type strain (Fig. 2B). These data show that the presence of the Rnq1 protein is required for the increased de novo prion formation observed in the ubc4 cells. To our knowledge, this is the first evidence demonstrating a role for the non-prion form of Rnq1 in the formation of a heterologous prion. In contrast, an antagonistic effect of ubc4 on [PSI+] curing by excess Hsp104 was detected in the [PSI+] rnq1 strain as clearly as in the [PSI+] RNQ1+ background (data not shown). This agrees with the previous observation that Rnq1 prion is involved in de novo [PSI+] formation but not in the maintenance of a pre-existing [PSI+] prion (19, 20).
Effects of UPS Alterations and Ssb Deficiency on Ub Levels and Antibiotic Sensitivity—We and others (32, 48) have previously observed that the ubp6 deletion results in depletion of free Ub and increased sensitivity to the translation inhibitor anisomycin and that most of the detectable phenotypes of ubp6 are due to Ub depletion. The proposed (30) involvement of Ssb in UPS-dependent protein degradation makes it tempting to speculate that some effects of ssb1/2 on [PSI+] could be through alteration of UPS. To examine potential mechanisms by which defects of UPS influence prion formation, we first characterized the levels of Ub and sensitivity to anisomycin in the strains with ubc4, ubp6, and ssb1/2 deletions. As expected, ubp6 reduced levels of free Ub in comparison with the isogenic wild-type strain (Fig. 3A). Interestingly, levels of free Ub were also reduced in the double ssb1/2 mutant (Fig. 3A). The ubc4 strain exhibited a slight increase in the levels of free Ub (Fig. 3A) apparently due to impaired conjugation. Deleting UBC4 in the ubp6 or ssb1/2 deletion strains (Fig. 3A) partially restored free Ub levels suggesting that the effects of UPS alterations on [PSI+] curing are not simply due to decreased Ub levels.
As shown previously for ubp6 (32), decreased Ub levels correlate with increased sensitivity to anisomycin. We found that like ubp6, both ubc4 and ssb1/2 mutants were more sensitive to anisomycin than the wild type strain. The ubp6 strain exhibited the highest level of sensitivity, the ubc4 strain exhibited an intermediate sensitivity, and ssb1/2 was the least sensitive. The double ubc4 ubp6 deletion strain was more sensitive to anisomycin then either single mutant, while the sensitivity of the triple ubc4 ssb1/2 deletion strain was closer to that of the ubc4 than to the ssb1/2 strain (Fig. 3B). These data indicate that decreased Ub levels and decreased Ub conjugation affect resistance to anisomycin in a partially independent fashion. Interactions between Ubc4, Ubp6, and Ssb affect both Ub levels and anisomycin sensitivity differently than interactions between the same players affect the [PSI+] curing assays. This suggests that the effects of UPS alterations on [PSI+] cannot be explained as a simple consequence of the alterations in Ub levels.
Ubiquitinated Sup35 Is Not Detectable in Vivo—The simplest explanation for the effects of UPS alterations on [PSI+] is that misfolded Sup35, generated either spontaneously or as a result of the Hsp104-mediated prion disaggregation, is normally eliminated via the Ubc4-mediated ubiquitination and subsequent proteasome-mediated degradation. If this process is defective in the UPS mutants, accumulation of misfolded Sup35 would lead to increased spontaneous prion formation and decreased prion curing. To address the possibility that misfolded Sup35 is targeted for UPS-mediated degradation via ubiquitination, we have searched for evidence of ubiquitinated Sup35 using two different approaches. First, we employed immunoprecipitation of Sup35 from cell lysates followed by immunoblotting with a Ub-specific antibody. These experiments were performed with both epitope-tagged Sup35-HA (Fig. 3C) and endogenous Sup35 (data not shown). Some experiments also employed the strains containing tagged derivatives of Ub (Ub-Myc in case of Sup35-HA (Fig. 3C) or Ub-HA in case of endogenous Sup35). Second (Fig. 3D), we purified Ub-protein conjugates from cell extracts using either a Ub conjugates enrichment kit, based on the Rad23 UBA domain fused to agarose beads, or protein A- and Myc-agarose for the HA-tagged or Myc-tagged Ub. The eluted Ub conjugates were then detected by Sup35-specific antibody. In both assays, wild-type strains were compared with the isogenic mutant strains in which Ub levels are depleted (that is, strains carrying doa4 or ubp6 mutations) or to the ubc4 strains where Sup35 ubiquitination was not expected to occur. As it is likely that ubiquitinated Sup35 is short-lived, some experiments were performed in the presence of an inhibitor of proteasomal proteolysis (MG132), using the specially constructed mutant strains (erg6 or pdr5) with increased permeability of the yeast membrane to MG132. In the other experiments, we employed the rpn4 mutant that exhibits decreased proteasomal function due to a defect in transcription of proteasome genes (49). All major experiments were repeated with a series of the isogenic strains that differed by prion composition (that is, presence or absence of [PSI+] and [PIN+]), as well as by levels of the Sup35 production (normal or high). Immunoprecipitation experiments were also performed with the [PSI+] strains overproducing Hsp104.
Despite numerous tries, none of the experiments produced any evidence for the existence of ubiquitinated Sup35. Neither were steady state levels of Sup35 influenced by the proteasome inhibitors (Fig. 3E). Likewise, a large scale analysis of the ubiquitinated proteins in yeast (50) failed to detect Sup35, although ubiquitinated forms of another prion protein (Rnq1) and some members of the Hsp70 family were detected. These negative results do not completely exclude the possibility that there is a small and short-lived ubiquitinated fraction of Sup35 that cannot be identified by the approaches used. However, at least in the case of the excess Hsp104-mediated [PSI+] curing, the ubiquitinated fraction of Sup35 should be sizeable for ubc4 to influence the outcome, and our results make such an explanation unlikely. Therefore, we propose that UPS alterations influence [PSI+] via a more complex mechanism than simply by altering Sup35 ubiquitination.
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ubc5 double deletion increases background levels of some stress-inducible chaperones, specifically Hsp70s (51). As both Sup35 levels and Hsp levels control [PSI+] propagation (12), one possibility could be that UPS alterations influence [PSI+] via alterations of either Sup35 or Hsp levels. To address this possibility, we checked the effects of UPS alterations on the levels of Sup35 and levels of Hsps known to influence [PSI+].
Our experiments detected no differences in Sup35 levels between the wild-type strain and isogenic strains bearing the single ubc4 and ubp6 or double ubc4 ubp6 deletions. This observation was confirmed for both [PSI+] (Fig. 4A) and [psi-] (data not shown) backgrounds. There was also no detectable change in the levels of Ssb, Hsp40-Ydj, or Hsp40-Sis1 chaperones in any of these strains (Fig. 4A). Level of Hsp104 and total Ssa protein were slightly but reproducibly increased by ubc4 in both [PSI+] (Fig. 4, A and B) and [psi-] (data not shown) backgrounds. This increase in Ssa levels was primarily due to induction of the normally silent SSA3 and SSA4 genes, as confirmed by using an Ssa3/4-specific antibody (data not shown). A slight but not statistically significant increase in Hsp104 levels was also detected in the ubp6 strain. However, neither Hsp104 nor Ssa levels were increased in the double ubc4 ubp6 mutant. The defect of [PSI+] curing by excess Hsp104 detected in this strain (see above), cannot be explained by alterations of the Hsp104 or Ssa levels.
Previously, we have shown that Sup35 and Ssa proteins physically interact in vivo (23). Interestingly, co-immunoprecipitation experiments demonstrated that the fraction of Ssa bound to Sup35 is increased about 3-fold in the ubc4 strain compared with the isogenic wild-type strain (Fig. 4, C and D), while total amount of Ssa in the cell is increased at only about 1.5-fold (Fig. 4B). These data indicate that lack of Ubc4 increases either ability of Ssa to recognize Sup35 or proportion of the Sup35 protein that can be recognized by Ssa or both. The possible relevance of this observation to the ubc4 effect on [PSI+] is discussed below (see "Discussion").
Aggregation Status of the Sup35 Protein in Strains with UPS Mutations—Next, we checked whether the aggregation status of the Sup35 protein is altered in strains with UPS defects. For this purpose, we have employed the Sup35NM-GFP construct that bears the N-proximal portion of Sup35 (encompassing the prion-forming and middle domains) fused to the green fluorescent protein. Expression of this construct from the copper-inducible (PCUP1) promoter results in accumulation of "clumped" or "dot"-like fluorescent aggregates in a fraction of the [PSI+] cells, while diffused fluorescence is detected in the [psi-] strains (52). We have observed that the [PSI+] ubc4 culture expressing Sup35NM-GFP contained larger proportion of cells with cytologically detectable aggregates, compared with the isogenic wild-type strain (Fig. 4E and data not shown). These results indicate that lack of Ubc4 facilitates formation of the large cytologically detectable Sup35 aggregates.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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ubp6 double mutant resembled that of the ubc4 single mutant. This indicates that both deletions likely influence prion curing via one and the same pathway. Most of the known phenotypic effects of ubp6 are explained by depletion of the free Ub pool (32, 48). This results in a decrease of Ub available for conjugation and consequently in a decreased amount of Ub-protein conjugates. Ubc4 is responsible for conjugating free Ub to the protein substrates, therefore ubc4 causes a more severe decrease in the amount of Ub-protein conjugates than ubp6 does, and the decrease of the Ub pool in a ubp6 strain cannot significantly add to this effect. Therefore, observation that ubp6 does not further exacerbate the effect of ubc4 on prion curing suggests that UPS effects on [PSI+] are mediated by the Ub-protein conjugation step. As Ubc4 is playing a major role in this step, we primarily focused on studying the effects of ubc4.
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also increased spontaneous [PSI+] formation. This is the first mutation shown to have such a "protein mutator" effect in the absence of the other pre-existing prion, [PIN+]. In principle, both effects of ubc4 on [PSI+] could be explained by a model, suggesting that misfolded Sup35 protein, occasionally generated spontaneously in the cells lacking prion or produced in result of disaggregating activity of Hsp104 on prion aggregates, is normally targeted by Ubc4-dependent ubiquitination for degradation via the proteasome. In the absence of Ubc4, this misfolded protein is not eliminated, increasing the probability of turning it into a prion. This model predicts that a fraction of Sup35 is ubiquitinated. Moreover, in order to explain the anti-curing effects of ubc4, the ubiquitinated fraction of Sup35 should be sizeable, at least in the prion-containing cells that overproduce Hsp104. However, neither direct approaches employed in our work (see above, Fig. 3, C and D) nor large scale analysis performed by another group (50) detected Sup35 among ubiquitinated proteins. Moreover, the prion domain of Sup35 contains a QN-rich stretch resembling poly(Q) proteins, and it is known that proteasomal degradation of proteins with poly(Q) stretches is inefficient (53). Although we cannot completely exclude a possibility that the small short-lived fraction of misfolded ubiquitinated Sup35 may exist in the yeast cells, it seems unlikely that elimination of such a fraction could explain all the observed effects of ubc4 on [PSI+].
Some UPS alterations are known to have broad pleiotropic effects, for example by inducing stress response. As some stress-induced proteins are shown to influence prions, we have checked whether levels of these proteins are altered in the cells with UPS alterations. Indeed, we have detected slightly increased levels of Hsp104 and Ssa in ubc4 and slightly increased level of Hsp104 in ubp6. However, neither of these chaperones was induced in the double ubc4 ubp6 mutant, where [PSI+] resistance to excess Hsp104 remained high. Moreover, neither overproduction of Hsp104 nor overproduction of Ssa leads to a detectable increase in spontaneous [PSI+] formation in the [psi- pin-] strain (data not shown). Therefore, it is highly unlikely that effects of UPS alterations on [PSI+] are explained by slight alterations in the levels of these chaperones. Remarkably, additive effects of ubc4 and ssb1/2 on [PSI+] curing by excess Hsp104 indicate that Hsp70-Ssb also influences prion resistance to excess Hsp104 via a pathway that does not involve Ubc4 and vice versa.
Interestingly, we have observed that while total levels of Ssa protein are only slightly increased in the ubc4 strain, the amount of Ssa bound to Sup35 is increased significantly. This could occur by one of the following two mechanisms: either 1) an increase in the affinity of Ssa to Sup35 or 2) an increase in the fraction of Sup35 that can be bound by Ssa. It is worth noting that these mechanisms are not mutually exclusive. The first mechanism is supported by the observation that Ssa was detected among ubiquitinated proteins (50). Thus, it cannot be excluded that its activity is somehow influenced by ubiquitination, which is defective in ubc4 strains. On the other hand, known patterns of Ssa interactions with other proteins speak in favor of the second mechanism. Indeed, Ssa with the help of the Hsp40 co-chaperones is known to bind partially unfolded proteins; therefore it is quite likely that it binds a partially misfolded fraction of Sup35. Increased Ssa binding to Sup35 in the absence of Ubc4 may therefore reflect either increased proportion of misfolded Sup35 in these conditions or its increased accessibility to Ssa.
Why, then, is misfolded Sup35 increased in abundance in the ubc4 cells if not due to lack of its Ub-promoted degradation? One possible explanation could be drawn from our observation that the fraction of cells containing large Sup35 aggregates is increased in the [PSI+] ubc4 culture (Fig. 4E). We have previously indicated (45) that large cytologically detectable aggregates of Sup35 share some (but not all) features of mammalian aggresomes, large cytoskeleton-associated structures generated by misfolded aggregation-prone proteins in the mammalian cells (54). It has previously been shown that aggresome formation is induced by UPS inhibition (55). It is therefore possible that defects of UPS, such as ubc4, induce an adaptive response in the yeast cells, which results in increased formation of the large aggresome-like structures. This response could be triggered by accumulation of the misfolded proteins due to defective Ub-conjugation but not necessarily by accumulation of misfolded Sup35 per se. In case of prion proteins, such aggresome-like structures promote segregation of the prion state from the action of chaperones and therefore contribute to increased resistance of prion state to the prion curing agents. In the non-prion cells, occasional "aggresome" formation by Sup35 initiates de novo prion generation.
Ssa protein has been implicated in disassembly of the large Sup35 aggregates into the smaller prion-propagating units (56), and our preliminary data suggest that Ssa can bind at least some cytologically detectable Sup35-GFP aggregates in the yeast cells.5 Therefore, increased formation of large aggregates may be responsible for increased Ssa binding to Sup35, for example by making Sup35 more accessible to Ssa.
Certainly this model does not explain all the complexity of UPS interactions with prions. Indeed, while favoring the [PSI+] state in curing assays, ubp6 decreased the phenotypic manifestations of [PSI+] and the de novo [PSI+] induction in the presence of excess Sup35 (32). These effects were even more pronounced in case of the deletion of DOA4, another gene whose product is involved in deubiquitination and maintenance of Ub pool. Interestingly, doa4 appeared to inhibit [PSI+] curing by excess Hsp104 as well, although this effect was difficult to characterize quantitatively due to severe growth defects caused by doa4 (data not shown). One possible explanation for the conflicting effects of ubp6 and doa4 on [PSI+] in different assays could be drawn from the necessity of ubiquitination for initiation of endocytosis (57), a process influencing formation of the yeast aggresome-like structures (45). Indeed, Doa4 is functionally linked to the endocytic pathway (58). Thus, ubp6, and especially doa4, may both induce Sup35 aggregation due to UPS defects as described above and inhibit it due to endocytosis defects caused by the depletion of free Ub pool. On the other hand, ubc4 is not predicted to affect endocytosis, as it does not deplete the free Ub pool and is not known to be involved in endocytosis-associated Ub conjugation.
Unexpectedly, we have found that ubc5 does not influence [PSI+] resistance to excess Hsp104 and de novo [PSI+] formation, despite the well known functional redundancy of Ubc4 and Ubc5 (51). One possible explanation is that modes of regulation of UBC4 and UBC5 are different from each other, so that Ubc4 is primarily expressed in the conditions that are crucially important for prion formation and curing. Indeed, some evidence suggests that Ubc4 and Ubc5 are preferentially produced at the different growth phases of the yeast culture (51). Such a hypothesis would indicate that processes of prion formation and elimination preferentially occur in certain physiological state(s), a notion that is worth investigating further.
At this moment, it is not known whether the Ub system specifically influences only [PSI+] or exhibits similar effects on the other prions. Although we have not detected any increase in de novo [PIN+] formation in the ubc4 background, one should note that we lacked an unbiased genetic system for [PIN+] detection, and therefore all we can say is that [PIN+]isnot being generated simultaneously with [PSI+]. While further experiments are needed to accurately address this question, our data have already provided one important insight into a role of the [PIN+] maintenance protein, Rnq1, in [PSI+] formation. Previously, it was shown that prion form of Rnq1 promotes de novo [PSI+] formation (18, 20). However, we have found that the presence of Rnq1 protein per se, even in its non-prion form, facilitates spontaneous [PSI+] formation at least in the ubc4 cells (Fig. 2B). This suggests that Rnq1 protein, independently of its aggregation, is playing an important role in the process of generation of other prions. Rnq1 was detected among ubiquitinated proteins in the large scale assays (50). It is therefore possible that some UPS effects on prions, e.g. increased spontaneous [PSI+] formation in ubc4, could be at least in part due to variations in Rnq1 ubiquitination and, consequently, its proteolyitic stability. This possibility is currently under investigation.
Taken together, our results present strong evidence for the ability of the UPS to regulate de novo prion formation and prion resistance to curing treatments in the yeast model. As the efficiency of ubiquitination and of proteasomal degradation may be influenced by various environmental and physiological conditions, this provides a potential new mechanism by which the cell may modulate protein assembly disorders and protein-based inheritance.
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This article was selected as a Paper of the Week.
1 These authors contributed equally to the paper.
2 Recipient of the Graduate Assistance in the Areas of National Needs (GAANN) fellowship from the Department of Education and by a Suddath Award from the Institute for Bioengineering and Bioscience, Georgia Institute of Technology. Present address: Center for Neurobiology and Behavior, Columbia University, New York, NY 10032.
3 To whom correspondence should be addressed: School of Biology, Georgia Institute of Technology M/C 0230, 310 Ferst Dr., Atlanta, GA 30332-0230. Tel.: 404-894-1157; Fax: 404-894-0519; E-mail: yury.chernoff{at}biology.gatech.edu.
4 The abbreviations used are: UPS, ubiquitin-proteasome system; Ub, ubiquitin; Ubc, Ub-conjugating enzyme; Ubp, Ub-specific processing protease(s); GFP, green fluorescent protein; GuHCl, guanidine hydrochloride; HA, hemagglutinin; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
5 R. Wegrzyn, L. Ozolins, and Y. Chernoff, unpublished data.
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Hsp104), and with the control plasmid pRS316GAL (Control). Transformants were selected on -Ura medium, replica-plated onto -Ura/Gal medium to induce the PGAL promoter, incubated for 4–5 days, and then velveteen replica-plated onto either complete YPD medium or -Ura-Ade medium. [PSI+] loss was scored by more reddish (dark on figure) color on YPD or decreased growth on -Ura-Ade. Levels of overexpressed Hsp104 were compared by Western blot using anti-Hsp104 antibody. B, epistatic interaction between ubc4
