‘ER degradation’ of a mutant yeast plasma membrane protein by the ubiquitin-proteasome pathway J.-M. GALAN, B. CANTEGRIT, C. GARNIER, O. NAMY,1 AND R. HAGUENAUER-TSAPIS2 Institut J. Monod, Universite´ Paris VII-CNRS, 2 place Jussieu, 75251 Paris Cedex 05, France

The yeast plasma membrane, uracil permease, undergoes ubiquitin-dependent endocytosis and subsequent degradation in the vacuole via a process that does not involve the proteasome. Cellsurface ubiquitination of this protein is mediated by the ubiquitin-protein ligase Npi1p/Rsp5p and involves Lys63-linked ubiquitin chains. This report describes the intracellular fate of a mutant form of uracil permease carrying a three amino acid insertion in a cytoplasmic loop. Most of this protein is not deployed beyond the ER, and is degraded by the 26S proteasome. Mutant permease degradation is almost unaffected in cells with impaired Npi1p/Rsp5p, but is dependent on the Ubc6p and Ubc7p ubiquitin-conjugating enzymes, suggesting that proteolysis of the protein requires its prior ubiquitination. Overproduction of a derivative of ubiquitin with a modified Lys48 strongly impairs mutant permease degradation. This suggests that, like other proteasome substrates, mutant permease might be polyubiquitinated with Lys48-linked ubiquitin chains. These findings provide an example of a yeast plasma membrane protein that is routed to the ‘ER degradation’ pathway, and highlight the versatility of the ubiquitin system.—Galan, J.-M., Cantegrit, B., Garnier, C., Namy, O., Haguenauer-Tsapis, R. ‘ER degradation’ of a mutant yeast plasma membrane protein by the ubiquitin-proteasome pathway. FASEB J. 12, 315–323 (1998) ABSTRACT

Key Words: proteasome · ubiquitin · uracil permease

THE ENDOPLASMIC RETICULUM (ER)3 is the point of entry of proteins into the secretory pathway. It is also where nascent secretory and membrane proteins are processed and acquire their tertiary and quaternary structures. It has long been known that quality control mechanisms in the ER ensure that nascent proteins that fail to fold correctly never reach the Golgi apparatus and are rapidly degraded. This process has been known as ‘ER degradation’, because target substrates had been localized to the ER region (1). Our understanding of the mechanisms involved in ER degradation has grown considerably in the past 2 years (reviewed in refs 2, 3). We now know that this degradation pathway does not target only misfolded

proteins, but is also involved in the turnover of certain ER resident proteins. Several groups have shown that various soluble and membrane-bound proteins retained in the ER are degraded in the cytoplasm by a multiprotease complex, the proteasome. Proteolytic breakdown may (4–6) or may not (7, 8) be preceded by polyubiquitination of the target proteins. Proteins must be transported back or ‘dislocated’ into the cytosol for cytoplasmic proteolysis to occur. Recent data indicate that the Sec61p complex, involved in translocation of proteins into the ER, also provides the conduit through which proteins are extruded back into the cytosol (9–11). The endoplasmic reticulum lumenal chaperone BiP (Kar2p) and membrane-bound Sec63p, other subunits of the import machinery in Saccharomyces cerevisiae, are also involved in the export of a misfolded protein to the cytosol (10). Some aspects of retrotranslocation may vary from one protein to another. The US2 protein of cytomegalovirus accompanies MHC class I molecules to the cytoplasm (11). Two genetic approaches to studying ER degradation in S. cerevisiae have revealed a small number of proteins that might be involved in recognizing misfolded proteins or in overall retrotranslocation (5, 12, 13). The ER-associated, membrane-bound protein Der1p is required before degradation of soluble CPY* and PrA*, which are mutant misfolded forms of the vacuolar carboxypeptidase Y (CPY) and proteinase A (PrA) (13). The membrane-bound protein Hrd3p acts at an early step before 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), an integral membrane protein of the ER that undergoes metabolically regulated degradation, is delivered to the proteasome (12). These two genetic approaches have converged and led to iden1

Present address: Institut Pasteur, 25–28 rue du Dr Roux, 75724 Paris Cedex 15, France. 2 Correspondence: Institut J. Monod, Universite´ Paris VIICNRS, 2 place Jussieu, 75251 Paris Cedex 05, France. E-mail: [email protected] 3 Abbreviations: ER, endoplasmic reticulum; CPY, carboxypeptidase Y; PrA, proteinase A; HMG-R, 3-hydroxy-3-methylglutaryl-CoA reductase; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CHX, cycloheximide; E2, ubiquitin-conjugating enzymes; E3, ubiquitin-protein ligases.

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tification of the ER resident Hrd1p/Der3p, which is essential to degradation of both soluble CPY* and the membrane-bound HMG-R (10, 12). Numerous examples of ER degradation have been found in mammalian cells (1), including cases of several human hereditary diseases such as Tay-Sachs disease, a1-antitrypsin deficiency, and cystic fibrosis (14). In contrast, few cases have been reported in the yeast S. cerevisiae, the known examples being CPY*, PrA* (5, 10), misfolded forms of a-factor (9) and of the ER-located Sec61p itself (4, 10), and normal degradation of the ER resident HMG-R (12). Extending the repertoire of substrates of the ER degradation pathway in yeast should help define the general and/ or specific features of this process. This report shows that a mutated form of the uracil permease from S. cerevisiae normally targeted to the plasma membrane undergoes ER degradation. We elucidated the topology of this protein by constructing mutant permeases carrying potential glycosylation sites on several hydrophilic loops of the protein (15). Although some variant permeases were correctly targeted to the plasma membrane and displayed uracil uptake activity, cells expressing one specific mutant protein had only residual permease activity. We have now shown that this mutant permease is not deployed beyond the ER, but degraded by the ubiquitin-proteasome pathway. This finding extends the list of ER degradation in yeast to a plasma membrane protein and also highlights specific questions about the components of the ubiquitin system involved in mutant permease recognition. Wild-type uracil permease is phosphorylated upon its arrival at the plasma membrane (16), and then undergoes cellsurface ubiquitination that involves the ubiquitin-ligase Npi1p (17). As for a few other yeast plasma membrane proteins (18), this posttranslational modification acts as a signal for endocytosis of the protein. It is subsequently degraded in the vacuole by a process that does not involve the proteasome (17). This is presumably because ubiquitin-permease conjugates are extended through ubiquitin Lys63 (19), unlike ubiquitin Lys48-extended chains, which are recognized by the proteasome (20). We therefore also determined whether mutant and wild-type permeases had similar or distinct requirements for various elements of the ubiquitin system.

MATERIALS AND METHODS Strains, plasmids, and growth conditions Strains used in this study are listed in Table 1. Plasmids YEp352fF (2 m URA3 FUR4) (17) and pgF (2 m LEU2 gal-FUR4) (16) carry the FUR4 gene under the control of its own promoter or the GAL10 promoter, respectively. Plasmids YEP352fF-430N and pgF-430N were constructed by in vitro mutagenesis and subcloning. Each is identical to 316

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TABLE 1. Yeast strains Strains

NC122 sp6 SL WCG 4a WCG4-11/22a MHY501 MHY623 MHY552 27061b 27064b

Genotype

Reference

MATa leu2 fur4D (XbaI1XbaI3) MATa sec18-1 leu 2-3, 2-112 MATa ura3 leu2-3, 112, his311,15 MATa ura3 leu2-3, 112 his311,15 pre1-1 pre2-2 MATa his3-D200 leu2-3,112 ura3-52 lys2-801 trp1-1 MATa his3-D200 leu2-3,112 ura3-52 lys2-801 trp 1-1 doa4D1::LEU2 MATa his3-D200 leu2-3, 112 ura3-52 lys2-801 trp1-1 ubc6D1::HIS3 ubc7::LEU2 MATa ura3 trp1 MATa npi1 ura3 trp1

27 16 33 33 44 44 37 17 17

YEp352fF or pgF, except they encode a mutant uracil permease (Fur4–430Np) carrying a three amino acid insertion that introduces a potential glycosylation site (NGT) after permease amino acid 429. YEp110 is a multicopy plasmid encoding a mutant version of a synthetic ubiquitin gene in which lysine 48 has been replaced by Arg (UbK48R) (21). This gene is placed under the control of the copper-inducible CUP1 promoter. Yeast strains were transformed according to Gietz et al. (22). For immunoblotting, yeast cells were grown in minimal medium (YNB) containing a 0.67% yeast nitrogen base without amino acids (Difco), supplemented with appropriate nutrients and 2% glucose as a carbon source for cells transformed with YEp352fF and YEp352fF-430N or 4% galactose plus 0.02% glucose for cells transformed with pgF or pgF430N. Overexpression of the CUP1 promoter was induced with 0.1 mM CuSO4. Chromosomal-encoded uracil permease is produced in very low levels and is undetectable by immunological techniques. Measurement of uracil uptake, preparation of yeast cell extracts, Western immunoblotting, cell fractionation, and equilibrium density centrifugation Uracil uptake was measured in exponentially growing cells as described previously (16). Cell extracts were prepared and proteins were analyzed by immunoblots, using an antiserum against the last 10 residues of uracil permease (23). Primary antibodies were detected by using a horseradish peroxidaseconjugated anti-rabbit IgG secondary antibody, followed by chemiluminescence (ECL, Amersham). The immunoblots were quantified by scanning densitometry, using NIH 1.59 software. Quantification was performed in the range where signal intensity was observed to be proportional to protein concentration. Cell organelles were fractionated on equilibrium density gradients, fractions were collected, and proteins were precipitated (24). The proteins in each fraction were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western immunoblotting for the presence of uracil permeases, Sss1p, an integral membranebound protein of the ER, and Pma1p, an integral membranebound protein of the plasma membrane (25, 26). The quantity of each protein in cell fractions was determined by scanning densitometry as described above.

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Figure 1. Topological model of uracil permease showing the position of the potential glycosylation site of mutant permease Fur4–430Np. Rectangles represent the putative transmembrane domains. Pulse-chase labeling and immunoprecipitation Yeast cells to be labeled were grown either in low-phosphate, low-sulfate medium (27) or in YNB (as indicated in the figure legends). They were labeled with [35S]methionine (Amersham) or [32P]orthophosphate (Amersham) (16). When needed, chase was performed by adding 10 mM cold methionine. Proteins were extracted, immunoprecipitated, and analyzed by Tricine SDS-PAGE using 10% resolving gels, as in ref 16. Dried gels were read with a PhosphorImager (Molecular Dynamics) and bands were quantified with ImageQuant software.

RESULTS A mutant uracil permease that fails to reach the plasma membrane The plasma membrane uracil permease, encoded by the FUR4 gene, has cytoplasmic amino and carboxyl termini (15). The orientation of several hydrophilic loops with respect to the membrane was investigated by glycosylation scanning and led to our proposal of a 10-transmembrane span topological model for the

protein (15). One of the mutant permeases constructed during these studies carried a potential glycosylation site (NGT) in a predicted cytoplasmic loop; the Asn part of the engineered glycosylation site followed permease residue 429 and thus was named Fur4–430Np (Fig. 1). This mutant permease was not glycosylated (28). Yeast cells that were disrupted for the FUR4 gene, but expressed this mutant permease, had only residual uracil uptake activity (5–10% of that in cells expressing the wild-type protein). This could result from a direct or indirect effect of the mutation on the permease active site or from a failure in permease targeting to the plasma membrane. We therefore checked the intracellular location of this mutant protein. Extracts from logarithmically growing cells expressing wild-type or mutant permeases were fractionated on sucrose density gradients in experimental conditions that give good separation of internal membranes from the more dense plasma membrane (24). The great majority of wild-type uracil permease was found, as expected, with the plasma membrane marker Pma1p, whereas a very minor fraction (at the limit of detection) was found with the internal membranes (Fig. 2A). In contrast, most of the mutant permease was found with the integral membrane-bound Sss1p (ER marker) (25) and only a little (5–10%) was found with the plasma membrane (Fig. 2B). As phosphorylation of Fur4p is a plasma membrane event (16), retention of the mutant permease in intracellular membranes is likely to affect its phosphorylation status. Cells expressing wild-type or mutant permease were pulse-chase labeled with [35S]methionine and labeled for 1 h with [32P]orthophosphate, in parallel (Fig. 3). Wild-type and mutant permeases were synthesized at the same rate, as revealed by 10 min labeling with [35S]methionine

Figure 2. Fur4–430Np lies in intracellular membranes. Cell lysates were prepared from 100 A600 of MHY501 cells transformed with YEp352fF (A) or YEp352fF-430N (B) grown in YNB medium with glucose as a carbon source. They were fractionated on 20–60% sucrose density gradients containing 10 mM EDTA. Aliquots of the various fractions were analyzed by Western immunoblots for uracil permease, and for Pma1p (plasma membrane marker) and Sss1p (ER marker). The relative amounts of the markers in each fraction (expressed as percent of the stronger signal) were measured by densitometry. The ER forms a characteristic double peak on these gradients (29). ‘ER DEGRADATION’ OF A YEAST PLASMA MEMBRANE PROTEIN

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Figure 3. Reduced phosphorylation of the mutant permease Fur4– 430Np. Nc122sp6 cells transformed with the plasmids pgF (2 m LEU2 galFUR4) and pgF-430N (2 m LEU2 galFUR4–430N) were grown to A600 Å 1 in low-phosphate, low-sulfate medium with galactose as a carbon source. They were labeled for 1 h with [32P]orthophosphate (lanes 1 and 4) or labeled for 10 min with [35S]methionine (lanes 2 and 5) and chased for 60 min with cold methionine (lanes 3 and 6). Protein extracts were prepared from 0.4 ml samples and incubated with anti-permease antiserum. The immunoprecipitates were analyzed by SDS-PAGE and fluorography. Phosphorylated permease species had a slightly lower electrophoretic mobility than [35S]methionine pulse-labeled permease (16).

(lanes 2 and 5). Labeling with [32P]orthophosphate gave a strong immunoprecipitated signal for wildtype protein, but the phosphorylated signal from cells expressing mutant permease was very faint (lane 4). Quantification of immunoprecipitated cpm indicated that the phosphorylation of mutated permease was reduced by at least 90% compared to wild-type protein for the same amount of [32P]cpm incorporated into total proteins. This experiment also showed that mutant permease was degraded after a 1 h chase (070%) (lane 6). Some degradation of wild-type protein, which occurs after its delivery to the plasma membrane (17), was also seen after a 1 h chase (lane 3). Introduction of three amino acids after residue 429 of uracil permease thus resulted in a mutant permease that did not reach the plasma membrane, and was retained in the intracellular membranes in a form susceptible to proteolytic degradation. Degradation of mutant permease occurs in a preGolgi compartment The cellular compartment in which proteolysis occurs was investigated using cells with a defective SEC18 gene. Sec18p, the analog of the mammalian N-ethylmaleimide-sensitive factor, is required for the fusion of ER-derived vesicles with the Golgi complex. The thermosensitive sec18–1 mutant grows normally at permissive temperature (257C), but the transport of soluble and membrane-bound proteins is blocked within minutes of a shift to a restrictive temperature (30). A pulse-chase experiment at 377C was performed on sec18–1 cells expressing wild-type or mutant permease (Fig. 4). The wild-type protein remained completely stable during the 2 h chase. Impairing the delivery to the Golgi of wild-type protein prevented its targeting to the plasma membrane, endocytosis, and subsequent degradation. In contrast, mutant permease was degraded with a t1/2 of Ç 30 min in the sec18–1 cells. Hence, proteolytic process318

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ing of mutant permease does not require transport beyond the ER. In agreement with this result, there was no significative difference in the rates at which mutant permease was degraded in wild-type and pep4 cells (data not shown), which are deficient in the activities of several vacuolar proteases and have a greatly reduced turnover of wild-type permease (17). Mutant permease degradation is dependent on the proteasome The few soluble and membrane-bound yeast proteins that undergo ER degradation are degraded by the 26S proteasome. This multisubunit complex is present in the nucleus and cytoplasm of all eukaryotic cells, and some particles are associated with the ER membrane (31). We investigated the role of the proteasome in the degradation of the mutant permease by using the double mutant pre1–1 pre2–2, whose chymotrypsin-like activity of the 20S proteasome core is impaired (32). PRE1 and PRE2 genes are both essential for viability. Cells carrying the mutations pre1–1 and pre2–2 degrade several short-lived proteins more slowly at 377C than do wild-type cells (32, 33). Pulsechase analyses were performed at 377C, using pre1–1 pre2–2 and isogenic wild-type cells expressing mutant permease. This protein was degraded much more slowly in pre1–1 pre2–2 than in wild-type cells (Fig. 5A). The half-life of the protein was twice as long in mutant (t1/2Å85 min) as in wild-type cells (t1/2Å40 min). There was also protection in pre1–1 pre2–2 cells when the degradation of mutant permease was followed by Western immunoblots after inhibition of protein synthesis by cycloheximide (CHX) (Fig. 5B). Quantification of the immunoblots also indicated a two- to threefold lower rate of mutant permease degradation in mutants compared to wild-type cells. The similarity of the results obtained by pulse-chase and CHX-chase experiments indicates that the degradation of mutant permease does not require protein synthesis. CHX-chase revealed that Fur4–430Np was degraded about twofold more slowly in thermosensitive cim3–1 and cim5–1 cells (data not shown), which are deficient in two 19S cap regulatory subunits of the proteasome (34). Normal function of Figure 4. Proteolysis of mutant permease in a pre-Golgi compartment. SL cells (sec18–1) (16) transformed with pgF or pgF-430N were grown at 247C in YNB medium with galactose as a carbon source. They were transferred to 377C for 10 min, labeled for 10 min with [35S]methionine, and chased for the times indicated. Protein extracts were prepared, immunoprecipitated, and analyzed as described in Fig. 3.

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Figure 5. The degradation of mutant permease is dependent on the proteasome. WCG4–11/22A (pre1–1 pre2–2) and isogenic parental WCG4a cells (WT) (33) were transformed with pgF-430N and grown to an A600 Å 0.8 at 307C in YNB medium plus galactose. Cells were transferred to 377C for 2 h. A) The turnover of mutant permease was measured by pulse-chase with [35S]methionine, as in Fig. 3. B) CHX (100 mg/ml) was added. Protein extracts were prepared at the times indicated and analyzed for permease by Western immunoblotting.

proteasome catalytic and regulatory subunits is therefore required for turnover of the mutant permease. Degradation of mutant permease is ubiquitin dependent The proteasome degrades ubiquitinated proteins and some nonubiquitinated proteins (31). We investigated whether ubiquitination is a prerequisite for the degradation of mutant permease. Ubiquitin molecules are transferred to lysine residues of target proteins via an E1-E2-E3 enzyme thioester cascade (ubiquitin-activating enzyme/ubiquitin-conjugating enzyme/ubiquitin-protein ligase). The yeast genome encodes 13 E2-related enzymes (35), including the two nonessential Ubc6p and Ubc7p, which are involved in the turnover of certain substrates of the ER degradation pathway. This was first revealed by genetic data (4, 5, 36). We compared the fate of mutant permease in wild-type and Dubc6 Dubc7 cells after inhibition of protein synthesis at 307C (Fig. 6). Mutant permease was degraded about half as fast in Dubc6 Dubc7 as in wild-type cells. The protection was further enhanced when degradation was followed at 377C (threefold difference in the turnover rates). These

data suggest that ubiquitination is required for normal degradation of mutant permease. Ubiquitin-dependent proteolysis requires polyubiquitination, which occurs by isopeptide linkage between the carboxyl terminus of one ubiquitin moiety and the internal Lys48 residue of the previously attached ubiquitin. The resulting polyubiquitin chains are recognized by human proteasome subunit 5a and its homologues in S. cerevisiae and A. Thaliana (20, 38, 39) in a manner highly cooperative with respect to chain length. Replacing ubiquitin Lys48 with Arg (UbK48R) prevents polyubiquitin chain formation and protein breakdown in vitro (40). Overproduction of UbK48R leads to stabilization of some ubiquitinated proteins in vivo (5, 41). But the protection has been found to be rather limited in several cases (21, 42, 43), probably because of the removal of UbK48R from multiubiquitin chains and its replacement by wild-type ubiquitin (42, 43). We used this mutant ubiquitin to test whether the formation of Lys48-linked ubiquitin chains is required for the degradation of mutant permease. Overproduction of UbK48R only slightly decreased the rate of degradation of the mutated permease in wild-type cells (data not shown). We therefore checked to determine whether overproduction of UbK48R might lead to stronger inhibition in cells having reduced cellular ubiquitin pools. We used cells lacking the Doa4p ubiquitin-isopeptidase that display defective proteolysis of several model substrates of the ubiquitin-proteasome pathway to variable degrees (44) and strong impairment in the ubiquitination of the cell-surface wild-type uracil permease (19). We have shown that ubiquitination of this permease is rescued in Ddoa4 cells by the overproduction of ubiquitin (19), as are several other abnormalities of Ddoa4 cells (S. Swaminathan and M. Hochstrasser, personal communication). These observations suggested that these cells are unable to maintain normal cellular ubiquitin pools. The degradation of mutant permease in Ddoa4 cells and wild-type cells was compared, a test that has not yet been reported for other substrates of the ER degradation pathway. The mutant permease was degraded at the same rate in a CHX experiment in both cases (Fig. 7A). This suggests that Ddoa4 cells have enough free ubiquitin for that particular process or that the Ubcs and/or ligases involved have greater

Figure 6. Effect of UBC6 and UBC7 deletions on the degradation of Fur4–430Np. MHY552 (Dubc6 Dubc7) (37) and parental MHY501 cells (WT) transformed with YEp352fF-430N were grown at 307C to A600 Å 0.7. CHX was added, and protein extracts were prepared at various intervals. Proteins were analyzed for mutant permease by Western immunoblots, as in Fig. 5B. Mutant permease appeared on SDS gels as one or two bands, depending on gel resolution. The lower mobility band was probably not phosphorylated, since it was insensitive to calf intestinal phosphatase (data not shown). ‘ER DEGRADATION’ OF A YEAST PLASMA MEMBRANE PROTEIN

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Figure 7. Overproduction of UbK48R stabilizes mutant permease against degradation. A) A CHX-chase experiment was performed as in Fig. 5B, using MHY501 (WT) and MHY623 (Ddoa4) transformed with YEp352fF-430N. B) MHY623 (Ddoa4) cotransformed with YEp352fF-430N and either YEp96 (encoding wild-type Ub) or YEp110 (encoding UbK48R) were grown with glucose as carbon source. CuSO4 was added for 2 h to induce the synthesis of ubiquitin. CHX was added, and the degradation of mutant permease was monitored as in Fig. 5B.

affinities for ubiquitin than that required for other ubiquitination events. The degradation of mutant permease was then followed after inhibition of protein synthesis in Ddoa4 cells overproducing either wild-type ubiquitin or UbK48R under the control of the copper-inducible CUP1 promoter. Overproduction of UbK48R strongly impaired the degradation of mutant permease that occurred normally upon overproduction of wild-type ubiquitin (Fig. 7B). This provides an independent argument that the proteolysis of mutant permease would be ubiquitin dependent and indicates that it probably requires polyubiquitination via Lys48-linked ubiquitin chains.

tent (17). As previously observed, wild-type permease was very slowly degraded in these mutant cells (1/4th the usual rate) (Fig. 8B). In contrast, mutant permease was degraded almost normally in cells carrying the npi1 mutation (Fig. 8A); the slight stabilization observed (õ1.5-fold) was not of the same order of magnitude as that of wild-type protein. Additional experiments performed at a restrictive temperature using rsp5–1 thermosensitive cells gave similar results (protection õ1.5-fold). This might result from stabilization of the small pool of residual plasma membrane Fur4–430Np. Thus, Npi1p/Rsp5p does not appear to be a critical element in the degradation of mutant permease.

DISCUSSION Several examples of ER degradation of yeast proteins have been described in the past few years. These include misfolded soluble proteins (5, 9) and membrane-bound proteins of the ER membrane, either misfolded (4) or undergoing normal regulatable degradation (12). This report describes an example of ER degradation of a yeast polytopic membrane protein that is normally targeted to the plasma membrane. A three amino acid insertion in a predicted cytoplasmic loop in the second half of the uracil permease of S. cerevisiae results in retention of the vast majority of the protein in the ER, where it is degraded. A small amount of this mutant protein escapes this process, reaches the plasma membrane, and yields uracil uptake activity. This situation is analogous to that of a mutant form of the human CFTR, in which the Phe at position 508 (D508) in a cytoplasmic loop of the protein is deleted. A tiny propor-

Degradation of mutant permease is barely affected in cells impaired in the ubiquitin-protein ligase Npi1p/Rsp5p The polyubiquitination of proteins depends on ubiquitin-conjugating enzymes (E2) and ubiquitin-protein ligases (E3). These ligases are generally believed to be the most directly involved in substrate recognition (35). To our knowledge, the E3 that takes part in the ubiquitination of substrates of the ER degradation pathway has not yet been identified in either yeast or mammals. On the other hand, the cell-surface ubiquitination of wild-type uracil permease that triggers its endocytosis depends on the Npi1p/Rsp5p ubiquitin-protein ligase (17). We therefore determined whether the essential Npi1p/Rsp5p was involved in the ubiquitination of Fur4–430Np, leading to its proteasome degradation. Mutant permease degradation was followed in parental cells and in npi1 cells, which have a greatly reduced Npi1p/Rsp5 con320

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Figure 8. Comparison of the effects of the ubiquitin-ligase Npi1p/Rsp5p on the degradation of wild-type and mutant permeases. A) 27061b (WT) and 27064b (pi1) were transformed with YEp352fF-430N. Cells were grown to A600 Å 0.7. CHX was added. Protein extracts were analyzed for uracil permease by Western immunoblots. B) The same experiment as in panel A was performed on cells transformed with YEp352fF (encoding WT permease).

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tion of mutant molecules reach the plasma membrane and give rise to chloride channel activity (14). The small pool of plasma membrane active mutant permease may be invaluable for the genetic screening of mutants in which the events targeting this protein for ER degradation would be impaired. The mutant permease could be located in the ER because of the fortuitous introduction of a specific ER retention signal or the impairment of an ER export signal. ER retrieval signals for soluble (KDEL/ HDEL) and bitopic (KK) membrane-bound proteins have been identified (45), as well as a diacidic signal required for the selective ER export of a type I transmembrane protein (46). However, the signals for ER retention and/or export of polytopic membranebound proteins remain ill-defined. The introduced mutation might also result from improper folding of the protein, as seems to be usual for substrates that undergo ER-associated degradation. Whatever the explanation, the observation that wild-type protein remains stable for hours in sec18 cells at restrictive temperature, whereas the mutant permease undergoes rapid, complete degradation, provides a striking illustration of the great specificity of sorting to the ER degradation pathway. As for the other substrates that undergo ER degradation, mutant permease proteolysis is independent of vacuolar proteases. It involves the proteasome, since it is impaired in cim3–1, cim5–1, and pre1–1 pre2–2 cells. The stabilization we observed is of the same order as reported in other cases (5). Since the protection is only two- to threefold, it is always possible that other unidentified proteolytic system (or systems) may contribute to the degradation process. Degradation by the 26S proteasome complex usually requires prior polyubiquitination of the proteolytic substrate, but not always (31). Several soluble and membrane-bound substrates of the ER degradation process undergo polyubiquitination (4–6), but some others, such as mammalian cytochrome P450 and 3-hydroxy-3-methylglutaryl-CoA reductase (7, 47) or a mutant form of the yeast pre-pro-a-factor (8), are not polyubiquitinated; the situation remains unclear in other cases (11). Deletion of the two UBC genes involved in the turnover of other yeast ER degradation substrates, UBC6 and UBC7, protects mutant permease against degradation, suggesting that this process requires prior ubiquitination of the protein. Ubiquitin-dependent endocytosis of wild-type permease is entirely independent of UBC6 and UBC7 (data not shown; and C. Volland, personal communication). The mutant permease is protected only incompletely in Dubc6 Dubc7 (two- to threefold), as for CPY* and PrA* (5). The limited effect on Fur4– 430Np might be due to the small pool of protein escaping to the plasma membrane or due to the participation of another of the numerous Ubcs in the

process. UBC4 has indeed been shown to be involved in the degradation of CPY* and PrA* (5). It is also possible that ER degradation of Fur4p-430Np involves both ubiquitin-dependent and independent processes. We used the mutant form of ubiquitin, UbK48R, to investigate whether mutant permease degradation requires polyubiquitination. This ubiquitin derivative can act as a terminator of Lys48-linked ubiquitin chain elongation. Overproducing this ubiquitin derivative in wild-type cells resulted in a slight stabilization of mutant permease. Overproduction of UbK48R in cells lacking the Doa4p ubiquitin-isopeptidase led to dramatic stabilization of mutant permease. We cannot rule out that this could possibly result from an indirect physiological effect of UbK48R in Ddoa4 cells, although we observed previously that this mutant ubiquitin is fully efficient for promoting Lys63linked ubiquitin chains extension of wild-type uracil permease in these cells. The strong stabilization of mutant permease by UbK48R in Ddoa4 cells suggests that polyubiquitination, probably involving ubiquitin chains linked through ubiquitin Lys48, is required before degradation of Fur4–430Np. The difference between the dominant effect of UbK48R in Ddoa4 cells and the weak protection it provides in wild-type cells could be due to the rapid removal of UbK48R from ubiquitinated mutant permease by the Doa4p isopeptidase in wild-type cells. On the other hand, the distinct effects of UbK48R might be due to a different balance between the pools of plasmid-encoded UbK48R and intracellular wild-type ubiquitin, enabling UbK48R to compete more efficiently with wildtype ubiquitin in Ddoa4 cells. Ubiquitin depletion is indeed thought to underlie some of the abnormalities of Ddoa4 cells (S. Swaminathan and M. Hochstrasser, personal communication), such as impaired ubiquitination of wild-type permease (19). The cell-surface ubiquitination of uracil permease is dependent on the Npi1p/Rsp5p ubiquitin-protein ligase. This protein contains a C2 domain (48) that mediates Ca2/-dependent attachment to phospholipids, which might be important for its involvement in membrane-associated ubiquitination. The degradation of Fur4–430Np is inhibited only very slightly in several npi1/rsp5 mutant cells, which all strongly stabilize wild-type protein. This residual protection might be due to stabilization of the small pool of Fur4–430Np escaping to the plasma membrane. Recent data indicate that uracil permease is phosphorylated before its cell-surface ubiquitination (49). The reduced phosphorylation of Fur4p-430Np might also explain why it is less susceptible to Npi1p/ Rsp5p-dependent degradation than is Fur4p. Although we cannot rule out some limited participation of this ligase in the ER-associated ubiquitination of mutant permease, this is probably no more than a side effect. This raises the question of the ubiquitin-

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ligase involved in ubiquitination of Fur4–430Np and other substrates of the ER degradation pathway. In addition to a formal demonstration that mutant permease is ubiquitinated, understanding the mechanisms involved in ER-associated degradation of the polytopic Fur4–430Np requires answers to many questions. For example, why does this particular mutation so specifically target the protein for ER degradation? Introducing the same three amino acids into a number of other loops of the permease does not trigger the same effect. The predicted cytoplasmic region 421–441 of uracil permease might interact with a particular chaperone. Little is known of the factors involved in the folding of this type of polytopic membrane-bound proteins in yeast. Perhaps cytoplasmic hsc/hsp70 assist folding of these proteins, as suggested for the mammalian CFTR (50). It is also possible that calnexin, which is involved in ER quality control of soluble and membrane-bound proteins in yeast and mammals (references in ref 51), interacts with Fur4p. Additional studies are required to elucidate the chaperones, DER and HRD gene products, and components of the translocon involved in the retrograde transport and ER-associated degradation of this mutant form of a plasma membrane protein. This study provides a suitable model system with which to pursue these components.

8.

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We thank M.-O. Blondel for in vitro characterization of the mutant permease. We are grateful to D. H. Wolf, C. Mann, M. Hochstrasser, J. Huibregtse, and B. Andre´ for generously providing strains and plasmids, and to R. Serrano and F. Kepes for the gift of antisera. We thank O. Parkes for editorial assistance. We are especially indebted to C. Volland, D. UrbanGrimal, and C. Marchal for numerous discussions and for critically reading the manuscript. This work was supported by a special grant from the CNRS (program ‘Biologie Cellulaire’, project #96105).

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Received for publication September 1, 1997. Accepted for publication October 30, 1997.

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