A Nonconserved Ala401 in the Yeast Rsp5 Ubiquitin Ligase Is Involved in Degradation of Gap1 Permease and Stress-Induced Abnormal Proteins

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A nonconserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins

Chikara Hoshikawa, Mika Shichiri, Shigeru Nakamori, and Hiroshi Takagi†

Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan

Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved July 29, 2003 (received for review May 23, 2003)

A toxic L-proline analogue, L-azetidine-2-carboxylic acid (AZC), AZC by acetylating it (14–17). It is also possible that a recessive causes misfolding of the proteins into which it is incorporated mutation occurs in the proline permease, the general amino acid competitively with L-proline, thereby inhibiting the growth of the permease encoded by GAP1, or the proline-specific permease cells. AZC enters budding yeast Saccharomyces cerevisiae cells encoded by PUT4 (18, 19); it may also occur in the membrane primarily through the general amino acid permease Gap1, not composition. through the proline-specific permease Put4. We isolated an AZC- AZC is a valuable compound for analyzing cellular response hypersensitive mutant that cannot grow even at low concentra- to protein misfolding, because substituting AZC in place of tions of AZC because of the accumulation of intracellular AZC. By L-proline in the protein will change the normal ␣-helical screening through a yeast genomic library, the mutant was found structure of a polypeptide to one having an angle of Ϸ15° to carry an allele of RSP5 encoding an E3 ubiquitin ligase. A single smaller, which results in an altered tertiary structure of the amino acid change replacing Ala (GCA) at position 401 with Glu protein (12). In this study, we isolated an AZC-hypersensitive (GAA) showed that Ala-401 in the third WW domain (a protein mutant that cannot grow on medium containing low concen- interaction module) is not conserved in the domain. The addition ؉ trations of AZC compared with its wild-type strain. We then of NH4 to yeast cells growing on L-proline induced rapid ubiquiti- identified the RSP5 gene encoding an E3 ubiquitin ligase nation, endocytosis, and vacuolar degradation of the plasma mem- involved in cell growth in the presence of AZC. The Rsp5 brane protein Gap1. However, immunoblot and permease assays protein is known to ubiquitinate plasma membrane permeases indicated that Gap1 in the rsp5 mutant remained stable and active followed by endocytosis and vacuolar degradation (20–22). In on the plasma membrane probably with no ubiquitination, leading the Rsp5 sequence of this mutant, one amino acid substitution to AZC accumulation and hypersensitivity. The rsp5 mutants also found in the nonconserved residue was shown to impair the showed hypersensitivity to various stresses (toxic amino acid specific interaction with the Gap1 permease. In addition, we analogues, high temperature in a rich medium, and oxidative propose a novel function of Rsp5 under various stresses that treatments) and defects in spore growth. These results suggest induce protein misfolding. that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogen- Materials and Methods regulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was Strains and Plasmids. All yeasts used in this study were the S. considered to have an important role in the ubiquitination of cerevisiae strains with a S288C background. CKY8 (MAT␣ targeted proteins. ura3-52 leu2-3,112) and ERX-17C (MATa his4-N met4 ade8) were supplied by C. Kaiser (Massachusetts Institute of Technol- ddition of some amino acid analogues can induce a transient ogy, Boston). An AZC-hypersensitive mutant CHT81, which has Aphysiological stress response in cells comparable to that of a mutation in RSP5, was isolated from CKY8 after ethyl heat shock stress (1–3). Most analogues are transported into cells methanesulfonate treatment. The rsp5 mutant INV-A401rsp5 via amino acid permeases and cause misfolding of the proteins (MATa his3-⌬1 leu2 trp1-289 ura3-52 rsp5::TRP1 p366- as they compete with naturally occurring amino acids. The A401rsp5[LEU2]) and its control strain INV-RSP5 (MATa accumulation of abnormal proteins, in turn, inhibits cell growth. his3-⌬1 leu2 trp1-289 ura3-52 rsp5::TRP1 p366-RSP5[LEU2]) Recently, Trotter and colleagues (4, 5) found that L-azetidine- were constructed from strain INVSc1 (MATa his3-⌬1 leu2 2-carboxylic acid (AZC), a toxic four-membered ring analogue trp1-289 ura3-52) (Invitrogen). Escherichia coli strain DH5␣ Ϫ␭Ϫ⌽ ⌬ ⌬ of L-proline, arrests proliferation in the G1 phase of the cell cycle [F 80lacZ M15 (lacZYA argF)U169 deoR recA1 endA1 Ϫ ϩ by the same mechanism as temperature up-shift. AZC is an hsdR17(rk mk ) supE44 thi-1 gyrA96] was used to subclone the unusual imino acid found only in several plants belonging to the yeast gene. Lilaceae family (6, 7), but can replace L-proline in proteins of A yeast genomic library was purchased from American Type bacteria and animal cells (8, 9), presumably in those of yeast cells Culture Collection (ATCC 77162). This library was constructed (5). When AZC was added to cells of yeast Saccharomyces by ligation of yeast genomic DNA partially digested with Sau3AI cerevisiae growing in minimal medium, cell viability gradually into the single BamHI site of YCp50-based p366. Centromere decreased, causing cell death (M. Nomura and H.T., unpublished plasmid p366 (ATCC 77163) was derived from YCp50 contain- work). ing CEN4 and LEU2 (11). Plasmid pCHT-1 containing RSP5 was The accumulation of abnormal or misfolded proteins in cells isolated from a suppressor of CHT81 by introducing a yeast under stress is a serious problem. To overcome it, the following genomic library. Plasmids p366-RSP5 and p366-A401rsp5 con- two strategies can be considered: (i) to degrade the proteins through a ubiquitin-proteasome system or (ii) to refold the proteins by molecular chaperones. We isolated AZC-resistant This paper was submitted directly (Track II) to the PNAS office. mutants and strains and elucidated the mechanisms of AZC Abbreviations: AZC, L-azetidine-2-carboxylic acid; YPD, yeast extract͞peptone͞dextrose. resistance. Large quantities of L-proline are believed to dilute Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. U18916, X52633, and M30583). AZC, its toxic competitor (10–13). We recently discovered the GENETICS MPR1 gene and its homologues on the chromosome of yeast †To whom correspondence should be addressed. E-mail: [email protected]. strains; these genes encode N-acetyltransferases that detoxify © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1933153100 PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11505–11510 Downloaded by guest on October 4, 2021 tains the wild-type and the Ala401Glu mutant RSP5, respec- integrated into the RSP5 locus in strain INVSc1 containing tively. Three centromere plasmids pRS414, pRS415, and pRS416 p366-A401rsp5 and p366-RSP5 to construct INV-A401rsp5 and (Stratagene) harboring TRP1, LEU2, and URA3, respectively, INV-RSP5, respectively, by transformation. The Trpϩ pheno- were used for complementing the auxotrophic markers. type was selected, and the correct disruption event was confirmed by using the chromosomal PCR. Culture Media. The media used for growth of S. cerevisiae were yeast extract͞peptone͞dextrose (YPD) (2% glucose, 1% yeast Northern Blot Analysis. Northern blot analysis was carried out by extract, 2% peptone) and SD (2% glucose, 0.67% Bacto Yeast using a Gene Images Random-Prime Labeling and Detection Nitrogen Base without ammonium sulfate and amino acids; system (Amersham Pharmacia). Total RNA from S. cerevisiae ϩ Difco). SD medium contained 10 mM (NH4)2SO4 (SD Am) or was isolated by the method of Ko¨hrer and Domdey (24). RNA 10 mM L-proline (SDϩPro) as the sole nitrogen source. Yeast was separated in 1.0% agarose gel and transferred to nylon strains were also cultured on SDϩAm agar plates containing membrane. As a DNA probe, the DNA fragments of GAP1, AZC, L-canavanine (L-arginine analogue), O-fluoro-DL- PUT4, and ACT1 were prepared by PCR with oligonucleotide phenylalanine (L-phenylalanine analogue), DL-␤-hydroxynorva- primers 5Ј-TTG ACG AAA CAG GTT CAG GG-3Ј,5Ј-TGC line (L-valine analogue), DL-norleucine (L-methionine ana- TGG GAT GAA AAG CTT CC-3Ј,5Ј-GAT TAT GGA CGT logue), ␤-(2-thienyl)- DL-alanine (L-alanine analogue) or S-2- GGA CTT GG-3Ј,5Ј-AAT GAG AGA GAA CCA CAC GG-3Ј, aminoethyl-L-cysteine (L-lysine analogue). All of the analogues 5Ј-CGG AAT TCC TCT CCC ATA ACC TCC TA-3Ј, and were obtained from Sigma. The E. coli recombinant strains were 5Ј-CGG GAT CCG GGC TCT GAA TCT TTC GT-3Ј, respec- grown in Luria–Bertani medium (23) containing ampicillin (50 tively. Each unique amplified band was purified from agarose ␮g͞ml). If necessary, 2% agar was added to solidify the medium. gel, denatured, and labeled according to the protocol recom- mended by the supplier. Gene Disruption. For GAP1 and PUT4 disruption, a DNA fragment containing URA3 and LEU2, respectively, was amplified by Western Blot Analysis. Yeast cells were cultured to the exponential ϩ PCR performed with genomic DNA from strain CKY8 and growth phase (OD600 of 1.0) in SD Pro medium, and the plasma oligonucleotide primers 5Ј-CGC TCT GGA TGA GAC ATA membrane-enriched fractions before addition of 10 mM TAA AGA TGA AGG TGA AGT CCA CTG ATT CGG TAA (NH4)2SO4 and at intervals thereafter were prepared as de- TCT CCG AAC-3Ј,5Ј-AAT CCC CAA AGT TCA ATA AGG scribed by Galan et al. (22). The cells were harvested and washed, AAA TGG GGA GTA AGG AGG CAT CAT TAC GTC CGA and the whole-cell extracts were prepared by vortexing the cells GAT TCC-3Ј,5Ј-GCA CAG CAT ATC TCC ATC CAT GTA with glass beads. The membrane-enriched fraction was collected CTG ATA CAG ACG CAT AGT CCT GTA CTT CCT TGT by centrifugation for 45 min at 15,000 ϫ g, suspended in 0.1 M TCA-3Ј, and 5Ј-TTA CCC CTG TTT CCT ATG CTG CGT Tris⅐HCl buffer (pH 7.5) containing 0.15 M NaCl, 5 mM EDTA, CGC TAA ATA AAA CCG ATC GCG CGT TTC GGT GAT and 5 M urea, kept on ice for 30 min, and sedimented as above. GAC-3Ј. The underlining indicates the sequence upstream of the The resulting pellets were resuspended in 0.1 M Tris⅐HCl buffer initiation codon and downstream of the termination codon of (pH 7.5) containing 0.15 M NaCl, 5 mM EDTA, and 2% SDS, GAP1 and PUT4, respectively. The unique 1.1- and 1.9-kb and boiled for 10 min. amplified band containing URA3 and LEU2, respectively, was For Western blot analysis, the supernatant (30 ␮g of solubi- purified and then integrated into the GAP1 and PUT4 locus, lized proteins) after centrifugation (10 min at 15,000 ϫ g) was respectively, in strain CKY8 by transformation. The Uraϩ and loaded on a 6% SDS-polyacrylamide gel in a Tricine system (25). Leuϩ phenotype was selected, and the correct disruption event For the experiment showing the shift of the ubiquitinated form was confirmed by using the chromosomal PCR. of Gap1, SDS͞PAGE was carried out on a 12.5% gel in Laemmli’s system (26). Gap1 was detected by using a ECL Plus Construction of the rsp5 Mutant. Aside from the mutant CHT81, Western Blotting Detection System (Amersham Pharmacia) and we constructed another rsp5 mutant INV-A401rsp5 by disrupt- anti-Gap1p polyclonal antibody at 1:30,000 dilution (a gift from ing an essential RSP5 gene on the chromosome of strain INVSc1 B. Andre´, Universite´ Libre de Bruxelles, Brussels). The plasma containing plasmid p366-A401rsp5. First, plasmid p366-RSP5 membrane Hϩ-ATPase Pma1 was used as an internal control. was constructed by blunt-end ligation of the 4.9-kb NcoI-AccIII Protein concentrations were determined with a BCA Protein fragment from pCHT-1 with the BamHI site of p366 and used Assay Reagent Kit (Pierce). as a template DNA for site-directed mutagenesis. The replacement of Ala-401 by Glu in Rsp5 was performed by PCR with Proline Permease Assays. Yeast cells were cultured to the expo- Ј ϩ oligonucleotide primers 5 -TAC GGA* ACG TGT ATA TTT nential growth phase (OD600 of 1.0) in SD Pro medium and CG-3Ј,5Ј-TAT ACA CGT T*CC GTA TTG GT-3Ј,5Ј-AAC proline permease activities were measured by incorporation TAC CTT CGT CAA GTC CG-3Ј, and 5Ј-ATC TTT CAT CGT of 14C-radiolabeled L-citrulline and L-proline (New England Ј CGG CAC TG-3 . The asterisks show the locations of mis- Nuclear) before addition of 10 mM (NH4)2SO4 and at intervals matches. The unique amplified band of 1.4-kb was digested with thereafter. The cells were harvested, washed, and suspended PvuII and AvaII to recover the 1.1-kb fragment and ligated to the in 10 mM citric acid buffer (pH 4.5) containing 2% glucose to 12.3-kb fragment of plasmid p366-RSP5 digested with PvuII and OD600 of 2.0. One milliliter of cell suspension was incubated AvaII. The mutation in the resultant plasmid p366-A401rsp5 was with 89 ␮M L-[14C]citrulline (56 mCi͞mmol; 1 Ci ϭ 37 GBq) confirmed by DNA sequencing. for Gap1 or with 22 ␮M L-[14C]proline (229 mCi͞mmol) for To disrupt RSP5 on the chromosome, a DNA fragment Put4 for 5 min at 25°C. The cells were then collected on a containing TRP1 was amplified by PCR performed with genomic 0.8-␮m nitrocellulose filter (Whatman) and washed twice with DNA from strain INVSc1 and oligonucleotide primers 5Ј-ATG ice-cold water. The filters were dried, and intracellular labeled CCT TCA TCC ATA TCC GTC AAG TTA GTG GCT GCA amino acid was determined by liquid scintillation counting. GAG TTA CTA TTA GCT GAA TTG CCA-3Ј and 5Ј-TCA Permease activities were calculated as nmol per min per mg TTC TTG ACC AAA CCC TAT GGT TTC TTC CAC GGC dry weight. CAA TGG TTT TTC GCC CTT TGA CGT-3Ј. The underlining indicates the sequence upstream of the initiation codon and Intracellular Content of AZC. Yeast cells were cultured in SDϩAm downstream of the termination codon of RSP5. The unique medium containing AZC, and 5 ml of cell suspension (Ϸ5 ϫ 108 1.5-kb amplified band containing RSP5 was purified and then cells) were removed, washed twice with 0.9% NaCl, and sus-

11506 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1933153100 Hoshikawa et al. Downloaded by guest on October 4, 2021 Fig. 1. AZC was mainly transported into yeast cells via Gap1. The growth phenotype and AZC content of each strain were examined. Approximately 106 cells of each strain and serial dilutions of 10Ϫ1 to 10Ϫ4 (from left to right) were spotted onto SDϩPro medium or SDϩAm plus AZC medium. The plates were incubated at 30°C for 3 days. The intracellular AZC content was measured 2 h after addition of 5 mM AZC in SDϩAm liquid medium. The values are means from three independent experiments. Variations in the values are Ͻ10%.

pended in 0.5 ml of distilled water. The 1.5-ml microcentrifuge found a single-base change from C to A at position 1,202, leading tube containing cells was transferred to a boiling water bath, and to replacement of alanine with glutamate at position 401 within intracellular amino acids were extracted by boiling for 10 min. the WW3 domain (Fig. 2B). It is noteworthy that Ala-401 is a After centrifugation (5 min at 15,000 ϫ g), each supernatant was nonconserved residue among the domains and that its function subsequently quantitated with an amino acid analyzer (L-8500A, is unknown. To examine the effect of one amino acid substitution Hitachi). The AZC content was expressed as a percentage of the (Ala401Glu) in the Rsp5 protein on AZC-hypersensitivity, we dry weight. constructed another rsp5 mutant INV-A401rsp5 as described in Materials and Methods. In agreement with the results obtained Results with mutant CHT81, AZC hypersensitivity in strain INV- The Toxic AZC Enters Yeast Cells Primarily Through Gap1. L-Proline 401rsp5 was clearly evident (Fig. 2A). These results suggest that enters yeast cells via two transporters, the Gap1 general amino an Ala401Glu mutation in Rsp5 impairs the specific interaction acid permease and the Put4 proline-specific permease. The gap1- with the targeted protein. and put4-disrupted strain failed to grow on a SDϩPro agar plate, whereas the wild-type and the single-gene disrupted strains used Gap1 in the Ala401Glu rsp5 Mutant Remains Stable and Active on the L-proline as the sole nitrogen source (Fig. 1). We examined Membrane. Because AZC is mainly incorporated into the cell via which of the two permeases was mainly involved in AZC uptake. Gap1 (Fig. 1), we examined the effect of Rsp5 on GAP1 Fig. 1 shows that the gap1-disrupted strain grew on the AZC- expression and Gap1 activity. Northern blot analysis showed containing medium and accumulated smaller amounts of intra- quantitatively similar amounts of GAP1 transcripts in strains cellular AZC than the wild-type and the put4-disrupted strains. CKY8 and CHT81 in the presence or absence of (NH4)2SO4 (Fig. These results indicate that toxic AZC is transported into cells 3A). Springael and Andre´ reported that the addition of ammo- primarily through Gap1. nium ions to yeast cells growing on L-proline as the sole nitrogen source induces rapid inactivation and degradation of Gap1 Isolation and Analysis of the AZC-Hypersensitive Mutant. To analyze through a process requiring Rsp5 (27). They showed that am- the gene involved in growth when yeast cells are exposed to monium ions cause internalization of Gap1 by endocytosis, AZC, we isolated mutant CHT81, which is incapable of growing on SDϩAm medium containing a low concentration of 0.1 mM AZC (10 ␮g͞ml). This low level of AZC does not inhibit growth of the parent strain CKY8 (Fig. 2A). In this mutant, the intracellular content of AZC at2hafteraddition of 5 mM AZC was much higher than that of the wild-type strain (1.74% vs. 0.40% of dry weight), probably because a larger amount of AZC had been transported into cells. A heterozygous diploid (CHT81 ϫ ERX-17C) crossed with a wild-type strain of the opposite mating type (ERX-17C) grew on 0.1 mM AZC- containing medium, indicating that the AZC-hypersensitive growth phenotype of the mutant was recessive (Fig. 2A). Al- though sporulation of diploid cells was detectable, no ascospore from the diploid grew on YPD plates.

The AZC-Hypersensitive Mutant Carried an Allele of the RSP5 Gene. We then transformed the mutant CHT81 with a yeast genomic library and incubated the transformants on LeuϪ agar plates containing 0.1 mM AZC at 30°C for several days. One plasmid (pCHT-1) was recovered from the only Leuϩ transformant that was resistant to AZC. When pCHT-1 was used to retransform strain CHT81, all of the Leuϩ transformants were found to grow on the AZC-containing medium, suggesting that the mutated gene resided in the plasmid (Fig. 2A). Fig. 2. The recessive mutation of RSP5 enhanced sensitivity to AZC. (A) Various strains were cultivated on SDϩAm medium containing 0.1 mM AZC The nucleotide sequence of the ends of the inserted DNA Ϸ at 30°C for 3 days. (B) Sequence alignment of Rsp5 WW domains. WW1, showed that pCHT-1 had an 12-kb fragment on chromosome amino acids 229–266. WW2, amino acids 331–368. WW3, amino acids

V. Subcloning of this region indicated that the gene complement 387– 424. The ﬁlled circles at the top indicate the highly conserved residues GENETICS to the AZC-hypersensitivity was RSP5, which encodes an E3 of the WW domain. The arrow at the bottom shows the mutation found in ubiquitin ligase. In the Rsp5 sequence of mutant CHT81, we this study.

Hoshikawa et al. PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11507 Downloaded by guest on October 4, 2021 Fig. 3. Gap1 in the rsp5 mutant remained active without ammonium- induced ubiquitination on the plasma membrane. (A) Total RNA (Ϸ50 ␮g) from strains CKY8 and CHT81 was hybridized to GAP1 and ACT1 probes. Cells were grown on SDϩPro medium before (time ϭ 0) and 60 min after Fig. 4. The rsp5 mutants showed hypersensitivity to stresses that induce addition of (NH4)2SO4. The GAP1 and ACT1 probes detected the 2.2-kb and protein misfolding. The growth phenotypes of various strains were exam- the 1.4-kb transcripts, respectively. (B) Immunoblot and permease activity ined. Approximately 106 cells of each strain and serial dilutions of 10Ϫ1 to of Gap1 in the membrane-enriched extracts prepared before (time ϭ 0) and 10Ϫ4 (from left to right) were spotted and incubated onto SDϩAm medium at several times after addition of (NH4)2SO4. Gap1 and Put4 activity was containing the toxic analogue at 30°C for 3 days (A), YPD or SDϩAm 14 14 measured by incorporation of C-citrulline and C-proline, respectively. medium at the indicated temperature for 3 days (B), and the H2O2- To determine the Put4 activity, we used the gap1-disrupted strains derived containing SD medium or SD medium after heat shock treatment (50°C,6h) form CKY8 and CHT81. Each activity before addition of (NH4)2SO4 is given at 30°C for 3 days (C). as 100%. (Lower) Levels of the plasma membrane Hϩ-ATPase Pma1 as a protein-loading control. (C) Immunoblot of Gap1 from the membrane- enriched extracts prepared before (time ϭ 0) and at several times after ammonium ions. Although we did not detect ubiquitin directly, addition of (NH4)2SO4. The positions of ubiquitinated forms are indicated these signals were assumed to be the ubiquitin-conjugated forms with dots. of Gap1 judging from the previous report (27). However, it seems likely that no apparent ubiquitination occurred in the rsp5 delivering it into the vacuole for degradation. To determine the mutant. These results suggest that the mutant Rsp5 fails to amount and the permease activity of Gap1 in the rsp5 mutant, recognize and to ubiquitinate Gap1 even in the presence of we carried out Western blot analysis using anti-Gap1 antibody ammonium ions. and permease assays using 14C-labeled amino acids. As shown in Fig. 3B, the wild-type Gap1 was removed from the plasma The Ala401Glu rsp5 Mutant Shows Hypersensitivity to Various membrane and its permease activity gradually decreased after Stresses. Regarding the Rsp5 function, in addition to ubiquiti- the addition of ammonium ions, as occurred in the previous nation of various permeases and transcriptional regulators (20– report (27). However, Gap1 in the rsp5 mutant CHT81 was 22, 29), it is known that a mutant has increased sensitivity to shown to remain stable and active on the plasma membrane with stresses such as cadmium and L-canavanine (the toxic L-arginine little degradation. analogue) even at permissive temperatures (30). We therefore We further examined the effect of Rsp5 on Put4 activity tested the growth phenotype of the rsp5 mutants CHT81 and measured by incorporation of [14C]proline. To eliminate the INV-A401rsp5 with various stresses that induce protein misfold- influence of Gap1 on L-proline uptake, the gap1-disrupted ing in the cell (Fig. 4). When yeast cells were exposed to stresses strains were constructed from strains CKY8 and CHT81. There such as toxic amino acid analogues (L-canavanine and O-fluoro- was no significant change in Put4 activity between two strains, DL-phenylalanine), high growth temperature (37°Cor39°C) even after the addition of ammonium ions (Fig. 3B). This result combined with a YPD medium, and oxidative stresses including suggests that Put4 is not a targeted protein of Rsp5. hydrogen peroxide and heat shock treatments, the rsp5 mutants were found to be much more sensitive to these stresses than the Gap1 in the Ala401Glu rsp5 Mutant Might Be Not Ubiquitinated in the wild-type strains CKY8 and INV-RSP5. Similar results were ␤ ؉ Presence of NH4 . Ubiquitin is generally believed to be used as a obtained in the cases of other toxic analogues [DL- - signal for the endocytosis of some cell surface proteins and their hydroxynorvaline (2 mg͞ml), DL-norleucine (2 mg͞ml), ␤-(2- subsequent degradation in the lysosome and vacuole (28). thienyl)- DL-alanine (10 ␮g͞ml), and S-2-aminoethyl-L-cysteine Therefore, ubiquitination of Gap1 was tested by Western blot (10 ␮g͞ml)], and ethanol treatment (8% vol͞vol) (data not analysis of membrane-enriched extracts (Fig. 3C). In the wild- shown). It is interesting that temperature sensitivity of the type strain CKY8, two minor bands were seen just above the mutant was observed only on a rich complex YPD medium, not major Gap1 signal at 5 and 10 min after the addition of on a synthetic minimal SDϩAm medium. These results suggest

11508 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1933153100 Hoshikawa et al. Downloaded by guest on October 4, 2021 that Rsp5 is required for selective degradation of abnormal a nonconserved residue and is situated in turn-like or polar proteins generated by these stresses and that Ala at position 401 region (48). The mutation at position 401 in Rsp5 is novel is important for its function. because each of the point mutations in conserved residues, including two Trp residues, two Tyr residues, one Phe residue, Discussion and one Pro residue (see asterisks in Fig. 2B), abolished Gap1 Permease Is a Primary Transporter for AZC. We found that the binding of WW domains to proline-rich peptides or resulted in toxic AZC enters yeast cells primarily via Gap1. Gap1 permease temperature-sensitive growth defects (49–51). Our results can transport all of the naturally occurring L-amino acids found suggest that position 401 in Rsp5 is important for ligand in proteins and related compounds, such as ornithine and binding, leading to subsequent ubiquitination of the targeted citrulline, and several D-amino acids and toxic amino acid protein. Replacement of Ala-401 by the large polar residue analogues (31). It is reasonable that gap1-disrupted strain was Glu may cause structural changes because of steric hindrance less sensitive to AZC than the wild-type and put4-disrupted or may impair the electrical environment with the adjacent strains (Fig. 1). However, intracellular AZC still remained even residue Arg-402 in the ligand-binding pocket. In the sequence in the gap1 and put4 double disruptant (0.14% of dry weight). It of Gap1, a PXY motif (Pro-201–Lys-202–Tyr-203) is present in was postulated that AZC is in part transported through another a small hydrophilic region based on the hydropathy profile permease(s), that take up L-proline, such as the structural (19), as a potential ligand for Rsp5. It should be possible that analogue of L-proline ␥-aminobutyric acid-specific permease the Gap1 PXY motif interacts with the WW3 domain of Rsp5 Uga4 (32) or through unidentified permease(s) (33), whose without binding to that of an Ala401Glu variant. function is similar to that of Gap1 or Put4. Rsp5 Is Considered to Participate in Selective Degradation of Stress- Rsp5 Causes Degradation of Gap1, but Put4 Is Not a Target of Rsp5. Induced Abnormal Proteins. Our results also suggest a novel Gap1 permease is inactivated by the addition of a rich nitrogen function of Rsp5 involved in selective degradation of stress- source (ammonia) to cells growing on the poor nitrogen source induced abnormal proteins. Yeast strains carrying the rsp5 (27). Regarding the transcriptional regulation of GAP1 in the mutation showed enhanced sensitivity to stresses, such as addi- S. cerevisiae strain with a S288C background, GAP1 was found tion of the amino acid analogues in a minimal medium, H2O2 and to express at high levels caused by the Nil1 transcriptional transient heat shock treatments, or a combination of a rich activator when ammonia was used as a nitrogen source (34). growth medium (YPD) and elevated temperature (37–39°C), by Analysis of GAP1 expression levels showed that the decrease accumulating abnormal misfolded cell proteins. The growth in Gap1 activity in the wild-type strain CKY8 was not caused phenotype of the rsp5 mutants at elevated temperatures is not a by a decrease in GAP1 transcription and that the GAP1 result of the instability of Rsp5, because they proved not to be transcription of the rsp5 mutant CHT81 is not affected by the temperature-sensitive in either a SD medium (Fig. 4) or a addition of ammonium ions (Fig. 3). These results indicate that synthetic complete medium (data not shown). Gap1 activity is regulated at the level of posttranslation. It should be noted that hypersensitivity of the rsp5 mutants to Helliwell et al. (35) recently found that overexpression of toxic amino acid analogues is attributable to two independent either Bul1 or Bul2, which has been identified as a protein that defects: one being the ammonia-induced inactivation of Gap1, binds to Rsp5 (36. 37), could confer AZC resistance caused by which presumably uptakes the analogues (Fig. 3), and the other the reduction of Gap1 and Put4 activity. In contrast, the bul1 the rapid degradation of misfolded proteins into which the and bul2 double disruptant was hypersensitive to AZC, be- analogues are incorporated. We further examined the effect of cause the activity of both permeases may be increased (35). GAP1 (data not shown). The gap1-disrupted mutant was still However, in the wild-type strain we used, it is unlikely that hypersensitive to these analogues, suggesting that the rsp5 mu- Put4 is down-controlled by Rsp5 activity after addition of tation also affects degradation of misfolded proteins. These ammonium ions (Fig. 3B). observations may support a role for Rsp5 in metabolism of misfolded and abnormal proteins. Ala-401 in the WW3 Domain Is Essential for in Vivo Function of Rsp5. Oxidative stress during exposure to H2O2 and to heat shock RSP5 is an essential gene in S. cerevisiae; Rsp5 activity was produces oxygen-free radicals and other reactive oxygen spe- recently shown to affect a variety of cellular events regulated cies that could attack vulnerable proteins containing iron by ubiquitination, including an early stage of cell surface sulfur centers (52). Ethanol can also disrupt protein folding by proteins endocytosis [Gap1 (27), Fur4 (22), Tat2 (38), Gal2 a mechanism distinct from that of AZC (5). In general, an (39), Zrt1 (40), and Ste2 (41)], mitochondrial inheritance (42), appropriate rate of protein synthesis is essential to produce activation of the Spt23 transcription factor (29), degradation properly folded proteins. At high temperatures in a rich growth of the large subunit of RNA polymerase II (43), and actin medium, it seems likely that the high rate of protein synthesis cytoskeleton dynamics (44). The amino acid sequence of Rsp5 disturbs the correct folding of nascent polypeptides and results reveals several domains: a C2 domain, which may bind mem- in the accumulation of denatured proteins. In S. cerevisiae, brane phospholipids in a calcium-regulated manner, three WW E2 ubiquitin-conjugating enzymes Ubc4 and Ubc5, the gene domains (WW1, WW2, and WW3), which are evolutionally expression of which is heat inducible, were known to mediate conserved protein interaction modules to determine the sub- selective degradation of L-canavanine-derived short-lived and strate specificity and are composed of 40 amino acids each that abnormal proteins (4, 53). Loss of Ubc4 and Ubc5 activity constitutes a hydrophobic pocket for binding proline-rich impairs cell growth and leads to inviability at elevated tem- ligands (PXY and PPXY motifs) in substrate proteins, and a peratures or in the presence of L-canavanine. It is also a matter HECT domain (homologous to E6-AP carboxyl terminus), of course that the yeast cytosolic proteasome is deeply involved which catalytically ligates ubiquitin in the process of target in stress-induced proteolysis in which L-canavanine- proteins degradation (45). WW domains are presumably lo- incorporated proteins are ubiquitinated and degraded for cell cated at cellular membranes and fold into three antiparallel survival (54). Our findings suggest that stress-induced abnor- ␤-sheets (46). In particular, the WW3 domain (amino acids mal proteins are selectively degraded through a process re- 387–424) and the WW2 domain are required for the essential quiring Rsp5 E3 ubiquitin ligase, but we do not yet demon-

in vivo function of Rsp5, and preferentially bind to peptides of strate this interesting event in vivo. Trotter et al. (5) have GENETICS consensus sequence (A͞P)PPPYE, as determined by phage- recently reported that misfolded proteins mediate heat shock display experiments (43, 47). Ala-401 in the WW3 domain is activation of heat shock factor and its target genes. To obtain

Hoshikawa et al. PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 ͉ 11509 Downloaded by guest on October 4, 2021 Spore Growth May also Require the Rsp5-Mediated Degradation. Kanda (30) reported that an rsp5-uby mutant had defects in sporulation of the diploid, suggesting that Rsp5 participates in sporulation via degradation of a specific group of proteins. The heterozygous RSP5͞rsp5 diploid sporulated, but none of the ascospore grew on YPD plates (data not shown). This result shows that Ala-401 in the Rsp5 protein is important for spore growth, although the sporulation process is normal. One possible mechanism is that Rsp5 participates in spore growth through degradation of specific or nonspecific protein(s) and through supplying amino acids for new protein synthesis in the cells. In summary, it is suggested that Rsp5 participates in selective Fig. 5. Proposed model for Rsp5-regulated proteolysis. Rsp5 is believed to degradation of stress-induced abnormal proteins and specific ubiquitinate Gap1 in an ammonium-induced manner followed by endocytosis proteins for spore growth in addition to nitrogen-regulated and vacuolar degradation (27). Various stress conditions probably lead to accu- degradation of Gap1 permease (Fig. 5). In this process, Ala-401 mulate abnormal cellular proteins. The addition of amino acid analogues is of Rsp5 plays a significant role in ubiquitination of targeted believed to cause misfolding of proteins into which it is incorporated. Exposure to proteins. We must further analyze the rsp5 mutants isolated here H2O2, heat shock, and ethanol denatures vulnerable proteins (5, 52). Cell growth by a combination of a rich medium and elevated temperature may disturb the to investigate the mechanism. This approach could be a useful correct folding of newly synthesized proteins. Our results suggest that Rsp5 is method for studying stress responses and for breeding novel involved in degradation of the targeted proteins in these cellular events through stress-resistant yeast strains. ubiquitination. Yeast strains carrying the rsp5 mutation show hypersensitivity to these stresses and defects in spore growth, presumably because of failure of We are grateful B. Andre´ and J.-O. De Craene for the generous gift of ubiquitination-triggered degradation of these proteins. antisera and for valuable discussions on this work. We also thank C. A. Kaiser for providing yeast strains and M. Wada and M. Takahashi for helpful comments. The technical assistance of M. Hisano of our labo- the evidence for our hypothesis, we now investigate whether ratory is greatly appreciated. This work was supported by a grant-in-aid the rsp5 mutant results in its stronger or more prolonged for scientific research from the Ministry of Education, Culture, Sports, activation in response to heat shock or ethanol treatment. Science and Technology of Japan (Grant 14037262 to H.T.).

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