©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Ubiquitination Mediated by the Npi1p/Rsp5p Ubiquitin-protein Ligase Is Required for Endocytosis of the Yeast Uracil Permease (*)

(Received for publication, December 21, 1995; and in revised form, January 31, 1996)

Jean Marc Galan (1) Violaine Moreau (2) Bruno Andre (3) Christiane Volland (1) Rosine Haguenauer-Tsapis (1)(§)

From the  (1)Institut Jacques Monod/CNRS, Université Paris7-Denis Diderot, 2, place Jussieu, 75251 Paris Cedex 05, France, (2)Institut de Biologie Moleculaire et Cellulaire/CNRS, 15, rue Descartes, 67084 Strasbourg, France, and (3)Laboratoire de Physiologie Cellulaire et de Génétique des Levures, Université Libre de Bruxelles, Campus plaine CP 244, Boulevard du Triomphe, B-1050 Bruxelles, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Uracil uptake by Saccharomyces cerevisiae is mediated by the FUR4-encoded uracil permease. This permease undergoes endocytosis and subsequent degradation in cells subjected to adverse conditions. The data presented here show that uracil permease also undergoes basal turnover under normal growth conditions. Both basal and induced turnover depend on the essential Npi1p/Rsp5p ubiquitin-protein ligase. Epitope-tagged ubiquitin variants have been used to show that uracil permease is ubiquitinated in vivo. The ubiquitin-permease conjugates that are readily demonstrated in wild type cells were barely detectable in npi1 mutant cells, indicating that uracil permease may be a physiological substrate of the Npi1p ubiquitin ligase. The lack of ubiquitination of the permease in npi1 cells resulted in an increase in active, i.e. plasma membrane-located, permease, suggesting that there is a direct relationship between ubiquitination and removal of the permease from the plasma membrane. The accumulation of ubiquitin-permease conjugates in thermosensitive act1 mutant cells, deficient in the internalization step of endocytosis is consistent with this idea. On the other hand, the degradation of uracil permease does not require a functional proteasome since the permease was not stabilized in either pre1 pre2 or cim3 and cim5 mutant cells that have impaired catalytic (pre) or regulatory (cim) proteasome subunits. In contrast, both basal and stress-stimulated turnover rates were greatly reduced in pep4 mutant cells having defective vacuolar protease activities. We therefore propose that ubiquitination of uracil permease acts as a signal for endocytosis of the protein that is subsequently degraded in the vacuole.


INTRODUCTION

Yeast cells have developed many regulated transport systems that respond sensitively to changes in the extracellular environment. Saccharomyces cerevisiae has several amino acid and sugar permeases that undergo down-regulation at both the transcriptional and post-translational levels when a preferred nutrient becomes available (1, 2) . Providing cells grown on a poor nitrogen source with NH(4) ions blocks expression of the general amino acid Gap1 permease and any existing Gap1 is rapidly inactivated(3) . This latter phenomenon, called nitrogen catabolite inactivation, has recently been shown to be accompanied by the proteolytic breakdown of the Gap1 permease(4) . The maltose transporter undergoes similar dual regulation, including proteolysis triggered by glucose under nitrogen starvation conditions(5) . The degradation of ITR1 encoded transporter is triggered by adding its own substrate, inositol(6) . Control of permease stability therefore appears to be a general process, which provides a rapid way of turning off the uptake of unneeded nutrients. The molecular mechanism responsible for switching between the stability and degradation of these plasma membrane proteins remains poorly understood.

We have used uracil permease to examine the degradative pathway of yeast permeases. This permease, encoded by the FUR4 gene, is delivered to the plasma membrane via the secretory pathway (7) and is phosphorylated on serine residues in a post-Golgi compartment, probably at the plasma membrane(8) . Permease is rapidly degraded under several adverse conditions, such as the approach of the stationary growth phase or the inhibition of protein synthesis. Its degradation follows its endocytosis, as it is dependent on the products of the END3 and END4 genes(9) , required for endocytosis of pheromone receptors(10) . The permease accumulates in the vacuole of growing pep4 mutant cells, deficient in the activities of the main vacuolar proteases(9) . This suggests that there is a constitutive turnover in addition to that triggered by stressful conditions.

Uracil permease contains a 9-residue sequence similar to the ``destruction box'' of mitotic cyclins. This box appears to be essential for the conjugation of ubiquitin (Ub) (^1)to cyclins and subsequent proteolysis (11, 12) . A permease having a point mutation (R294A) within this region is resistant to stress-induced degradation(13) . We have therefore suggested that ubiquitination is a signal for permease degradation, as it is for the breakdown of the mitotic cyclins. Ub-conjugates formation requires the combined action of three classes of enzymes: the Ub-activating enzyme (E1), Ub-conjugating enzymes (UBC, or E2), and at least in some cases Ub-protein ligases (E3), which play a role in substrate recognition(14, 15) . The hypothesis that Ub is conjugated to the uracil permease is supported by the finding that the permease degradation triggered by stressful conditions requires the NPI1 gene product(4) , which is structurally and functionally related to E6-AP, a human Ub-protein ligase(4, 16) .

The data presented here demonstrate that uracil permease indeed undergoes Npi1p-dependent ubiquitination. We show that permease ubiquitination is a cell surface event, followed by endocytosis and vacuolar proteolytic breakdown. We therefore propose that ubiquitination of uracil permease signals its endocytosis.


MATERIALS AND METHODS

Strains, Plasmids, and Growth Conditions

The S. cerevisiae strains used were 23344C (MATalpha ura3), 27061b (MATaura3 trp1), 27038a (MATanpi1 ura3), and 27064b (MATanpi1 ura3 trp1), which are all derived from the wild type strain S1278b(17) ; NY279 (MATaura3-52, act1-3) (carrying in ACT1 gene a single point mutation identical to that present in act1-1) (18) and isogenic parental strain NY13 (MATaura3-52)(19) ; IW-6A (MATalpha pep4-3 ura3-1 his3-11 leu2-3, 112 trp1) (9) and its parental strain W303-1B/D (MATalpha ade2-1 ura3-1 his3-11 leu2-3, 112 trp1)(20) ; WCG4-11/22a (MATaura3 leu2-3, 112 his3-11, 15 pre1-1 pre2-2) and isogenic wild type strain WCG4a (MATaura3 leu2-3, 112 his3-11, 15) (21) ; CMY 763 (MATalpha cim3-1 ura3-52 leu2-Delta1 his3-Delta200), CMY 765 (MATalpha cim5-1ura3-52 leu2-Delta1 his3-Delta200), and congenic wild type strain YPH 499 (MATaura3-52 leu2-Delta1 his3-Delta200 trp1-Delta63 lys2-801 ade2-101)(22) . As the chromosome-encoded uracil permease is produced in very low concentrations, cells that expressed permease from 2 µ-based multicopy plasmids were used for the immunodetection of the permease. The plasmid pfF (2µ LEU2 FUR4) (8) carries the FUR4 gene under the control of its endogenous promoter, as does plasmid YEp352fF (2µ URA3 FUR4), which was constructed by inserting into the vector YEp352 (23) a 2.6-kilobase pair HindIII-BamHI fragment, carrying the FUR4 gene, derived from the plasmid pfF. In plasmids 195gF (2µ URA3 GalFUR4) and pgF (2µ LEU2 GalFUR4) (8) the FUR4 gene is under the control of the GAL10 promoter. The plasmid YEp96 (2µ TRP1 Ub) contains a synthetic yeast Ub gene under the control of the copper-inducible CUP1 promoter, YEp112 (2µ TRP1 HA-Ub) and YEp105 (2µ TRP1 Myc-Ub) are identical to YEp96, except that they encode hemagglutinin (HA) or c-Myc-tagged versions of Ub(24) . The derivative of pUb23 (2µ URA3 GalUb-lacZ) encoding a chimeric protein, in which the yeast Ub gene is fused with a modified beta-galactosidase carrying a proline residue instead of the initiator methionine(25) , will be referred to as p(Ub-Pro-beta-gal). Yeast strains were transformed according to (26) . Cells were grown at 30 °C (or 24 °C for thermosensitive strains) in minimal medium containing 0.67% yeast nitrogen base without amino acids (Difco) supplemented with appropriate nutrients. Carbon sources were 2% glucose, or 4% galactose plus 0.02% glucose (or 0.05% glucose in case of weakly gal strains). Media contained 0.1 mM CuSO(4) for experiments involving overexpression of Ub. Uracil uptake was measured in exponentially growing cells as described previously(8) .

Pulse-chase Labeling and Immunoprecipitation

Yeast cells transformed with multicopy plasmids encoding the FUR4 gene under the control of the GAL10 promoter were grown in YNB medium with galactose as a carbon source to an A of 0.7 (1.4 times 10^7 cells/ml). For pulse-chase labeling in growing cells, cells were labeled for 10 min by adding 50 µCi of [S]methionine/ml of culture and chased in 10 mM methionine. For pulse-chase labeling in stress conditions, cells were labeled with TranS-label for 10 min and chased in 10 mM methionine plus 10 mM cysteine. We have observed that the turnover rate of uracil permease is accelerated in the presence of cysteine, as it is after inhibition of protein synthesis by cycloheximide. Aliquots of the culture (0.5 ml) were removed at various times during the chase, and proteins were processed for immunoprecipitation as described previously (8) , using an antiserum raised against a peptide corresponding to uracil permease residues 15-30 (N-terminal antipeptide antibody). For immunoprecipitation of control Ub-Pro-beta-gal using anti-beta-galactosidase antiserum (Sigma), heating steps were performed for 4 min at 95 °C, instead of 10 min at 37 °C for uracil permease. Immunoprecipitated proteins were analyzed by electrophoresis in a Tricine system and fluorography as described previously(8) . The protein bands were quantified using a PhosphorImager (Molecular Dynamics) and the ImageQuant software package.

Yeast Cell Extracts and Western Immunoblotting

Cell extracts were prepared and proteins were analyzed by immunoblots as described previously (9) using an antiserum to the last 10 residues of the permease (C-terminal antipeptide antiserum), or, when indicated, with N-terminal antipeptide antibody, both affinity-purified(9) . Some immunoblots were also probed with an antiserum to the plasma membrane (H)-ATPase, used without further purification. Primary antibodies were detected with a horseradish peroxidase-conjugated anti-rabbit IgG second antibody followed by ECL chemiluminescence (Amersham Corp).

Membrane Preparation

Yeast cells in the exponential growth phase were harvested by centrifugation in the presence of 10 mM sodium azide, washed once in distilled water plus 10 mM sodium azide, and suspended in lysis buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 5 mM EDTA, and a mixture of proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluoride and 2 µg/ml each of leupeptin, aprotinin, pepstatin, and chymostatin) at a concentration of 50 A/0.2 ml in a 1.5-ml conical tube. All subsequent steps were carried at 4 °C. Chilled glass beads (0.45 µm diameter) were added to the meniscus, and the cells were lysed by vigorous vortex mixing for 3 min. The resulting homogenate was diluted 3-fold with the same buffer and centrifuged at 3000 rpm for 3 min to remove unbroken cells and large debris. The plasma membrane-enriched fraction was collected by centrifugation for 45 min at 12,000 rpm. This membrane pellet containing uracil permease was suspended in lysis buffer plus 5 M urea, kept at 0 °C for 30 min, and sedimented as above. The resulting pellets were resuspended in lysis buffer, and trichloroacetic acid was added to 10% to precipitate proteins. This step was needed to prevent hydrolysis by residual endogenous proteases. The precipitates were neutralized and dissolved in 20 µl of 1 M Tris base plus 80 µl of 2 times sample buffer (100 mM Tris-HCl, pH 6.8, 4 mM EDTA, 4% SDS, 20% glycerol, 0.02% bromphenol blue) containing 2% 2-mercaptoethanol and heated at 37 °C for 15 min. Membrane-bound proteins were either analyzed by Western blot as described above for total cell extracts, or immunoprecipitated with antibodies to uracil permease. The 2-mercaptoethanol was omitted from samples for immunoprecipitation; the samples were diluted with 0.6 ml of TNET buffer (TNET = 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) plus the mixture of proteinase inhibitors used above for membrane preparation. Immunoprecipitates were then analyzed by Western immunoblot with monoclonal antibodies, either anti-HA (ascitic fluid containing the 12CA5 antibody) or anti-c-Myc (purified 9E10 antibodies purchased from Santa Cruz Biotechnology) and a horseradish peroxidase-conjugated anti-mouse IgG second antibody, followed by ECL chemiluminescence (Amersham).


RESULTS

Npi1p-dependent Turnover of Uracil Permease in Exponentially Growing Cells

The npi1 mutation was originally isolated because of its ability to prevent the nitrogen catabolite inactivation of several nitrogen permeases, including the general amino acid permease Gap1p(27) . NPI1, also known as RSP5(16) or MDP1, (^2)is an essential gene. In a viable npi1 mutant carrying reduced amounts of NPI1 transcripts, the ammonium-induced degradation of Gap1p is strongly impaired(4) . The stress-induced degradation of uracil permease was also impaired in this mutant(4) . In order to find out whether Npi1p is also involved in the fate of uracil permease in actively growing cells, the half-life of uracil permease was determined by pulse-chase experiment in wild type and isogenic npi1 cells transformed with multicopy plasmids carrying the FUR4 gene under the control of the GAL10 promoter. Cells grown to mid-exponential growth phase were labeled with [S]methionine and chased with an excess of cold methionine. Uracil permease was then immunoprecipitated and analyzed by polyacrylamide gel electrophoresis, and the signals were quantified (Fig. 1). Uracil permease was synthesized at the same rate in wild type and npi1 mutant strains and a decrease in electrophoretic mobility appeared after a 30-min chase in both strains. This mobility shift resulted from the phosphorylation of the permease that occurred at the plasma membrane(8) . After a lag (30-60 min), permease was degraded with first order kinetics. The half-life of permease from this point was about 40 min in wild type cells, versus 80 min in npi1 mutant cells. As described previously, adding glucose to the medium of galactose-grown cells slowed down permease turnover. In this case, too, the npi1 mutation resulted in a 2-fold decrease in permease turnover rate. Hence, uracil permease appeared to be a moderately short-lived protein in growing cells, and Npi1p is involved in this basal turnover.


Figure 1: Stabilization of uracil permease in npi1 mutant cells. 23344C (WT) and 27038a (npi1) strains transformed with p195gF were grown to an A of 0.7 with galactose as a carbon source. Cells were then labeled by incubation for 10 min with [S]methionine in the growth medium and chased with methionine for the indicated times (in min). A, uracil permease was immunoprecipitated and analyzed by SDS-PAGE and fluorography. B, PhosphorImager analysis quantitation of this gel and other gels from independent experiments. bullet, wild type; circle, npi1 cells.



Accumulation of Uracil Permease at the Cell Surface in npi1 Mutant Cells

The reduced rate of permease degradation in npi1 cells resulted in a striking difference in the steady-state abundance of uracil permease in cells collected during growth. The amounts of immunodetectable permease in glucose-grown cells expressing permease under the control of its endogenous promoter were markedly greater in npi1 mutant cells than in parental cells (Fig. 2A). The concentration of permease in npi1 cells was 2 to 3-fold higher, as estimated by Western blot analysis of serial dilutions of npi1 extracts (not shown). In contrast, the amounts of a major plasma membrane protein, (H)-ATPase, reported to be a long-lived protein (half-life over 10 h) (28) were similar in parental and npi1 cells. There was also a two-fold higher uracil uptake throughout the growth for npi1 cells than for wild type cells (Fig. 2B). Thus, the protection against degradation of the uracil permease in npi1 growing cells seemed to be mainly due to its stabilization at the plasma membrane. A similar stabilization of active uracil permease was observed when npi1 cells overexpressing uracil permease were submitted to inhibition of protein synthesis(4) . The loss of uracil uptake triggered by inhibition of protein synthesis was also largely prevented at the restrictive temperature in a mdp1 (npi1) thermosensitive strain (29) expressing only chromosomal encoded uracil permease (data not shown). Therefore, whatever the level of expression of uracil permease, an Npi1p-dependent step seems to be required to remove the permease from the plasma membrane, or at least in an early step of the endocytic pathway.


Figure 2: Steady-state levels of uracil permease in growing wild type and npi1 cells. A, 23344C (WT) and 27038a (npi1) cells harboring YEp352fF were grown to an A of 0.7 (lanes 1 and 2) or 1.3 (lanes 3 and 4). Protein extracts were prepared, resolved by electrophoresis, and blotted onto a nitrocellulose filter. The blot was probed with both uracil permease and plasma membrane (H)-ATPase antibodies. The molecular masses of the markers are given in kDa. Permease appears as a broad band corresponding to different phosphorylated bands(8) . The resolution of these different species vary depending on the gels. B, uracil uptake activity (nmol/min/A) of the same cells measured during the growth phase. bullet, wild type; circle, npi1.



Npi1p-dependent Ubiquitination of Uracil Permease

Molecular cloning of the NPI1 gene showed that it encodes Rsp5p, a protein structurally related to the human E6-AP Ub-protein ligase(4) . The involvement of Npi1p in destabilizing the uracil permease strongly suggested that ubiquitination of the permease could be required for its degradation. Overexposed immunoblots often showed a pattern of low mobility species reminiscent of a ladder of Ub-conjugate bands. Epitope-tagged variants of Ub, overexpressed from the copper inducible CUP1 promoter(24) , were used to test whether these bands were indeed ubiquitinated forms of uracil permease. These tagged versions of Ub, larger than normal Ub, lead to a decrease in the electrophoretic mobility of ubiquitinated proteins. As the concentration of putative ubiquitinated permease species were low, we made use of plasma membrane-enriched fractions from cells that overexpressed uracil permease with or without tagged or untagged Ub. Fig. 3A shows an immunoblot probed with anti-uracil permease antibodies. All the detected bands corresponded to permease species, as no signal was seen in samples from control cells that did not overexpress uracil permease (lanes 6 and 7). Membranes from cells that overexpressed normal Ub (lane 3) showed, in addition to the main permease band, two minor bands, which appeared to be up-shifted upon overexpression of HA- or Myc-Ub, gaving rise to species with apparent molecular masses of 80 and 68 kDa (lanes 2 and 4). The nature of these slow-migrating species, which most probably represent gradually ubiquitinated forms of uracil permease, was further examined by immunoprecipitation of these enriched-plasma membrane fractions with antibodies to uracil permease. The precipitates were analyzed by Western blotting with monoclonal antibodies to HA or to Myc epitopes (Fig. 3B). No specific signal was seen in samples from cells that overexpressed only tagged Ub (lanes 2 and 5) or uracil permease and normal Ub (lanes 1 and 4). A species migrating just below the 84-kDa marker was clearly recognized by both HA and Myc antibodies in samples from cells overexpressing both permease and tagged Ub (lanes 3 and 6). This ``80-kDa'' species thus represents a Ub-conjugate that is presumed, owing to its size, to be a doubly ubiquitinated uracil permease. The ``68-kDa'' species, which seemed to be shifted into a slower-migrating form in the presence of tagged-Ub (Fig. 3A, compare lanes 2 and 4 with lane 3), did not yield detectable HA or Myc immunoreactivity. However, it might be a mono-ubiquitinated species that was below the limit of detection by our monoclonal antibodies. We conclude from these experiments that uracil permease is a target for ubiquitination in normal growing cells. In addition, we observed that the steady-state abundance of Ub-permease conjugates, and most notably that of the ``80-kDa'' species, increased upon overexpression of Ub. This increase was quite clear when comparing the amount of MycUb-permease conjugates observed with or without copper induction of MycUb. Similar observations were already reported in a few cases(24, 30) . This might indicate that the pool of Ub is rate-limiting for the formation of Ub-conjugates in some experimental conditions.


Figure 3: Ubiquitination of uracil permease. A, 27061b cells bearing plasmids p195gF (lane 1), YEp105 (lane 6), or YEp112 (lane 7) or both p195gF and either YEp112 (lane 2), YEp96 (lane 3), or YEp105 (lanes 4 and 5) were grown in the presence of CuSO(4) (except for lane 5), to induce normal or tagged Ub synthesis from the CUP1 promoter. + above the lanes indicates the overexpression of the corresponding proteins; u corresponds to a low production of Myc-tagged Ub from the uninduced CUP1 promoter. Crude membranes were prepared from cells collected in mid-log phase and analyzed by Western blot with uracil permease-specific antibodies. The molecular masses of the markers are given in kDa. B, membrane extracts were precipitated with anti-uracil permease antibodies and further analyzed by Western blot with mouse anti-HA (lanes 1-3) or anti-Myc (lanes 4-7) monoclonal antibodies. The indications above the lanes are as in A.



The Ub-permease conjugates in npi1 mutant cells and wild type cells were compared by Western blot analysis of plasma membrane-enriched fractions (Fig. 4). The high molecular mass species visible in parental cells (lane 1), identified as Ub-conjugates, were essentially absent from membrane-extracts from npi1 cells (lane 2). Even membranes derived from npi1 cells overexpressing HA-tagged Ub, which led to an enrichment of ubiquitinated species in parental cells (lane 3), showed no high molecular mass species (lane 4). Glucose-grown npi1 cells, which overexpressed uracil permease from its endogenous promoter, also lacked Ub-permease conjugates (not shown). It is therefore very probable that Npi1p is directly involved in the normal ubiquitination of the permease and thus contributes to its instability.


Figure 4: Lack of Ub-permease conjugates in npi1 cells. 23344C (WT) and 27038a (npi1) cells transformed with p195gF (lanes 1 and 2); 27061b and 27064b (their trp1 counterparts) cotransformed with p195gF and YEp112 (lanes 3 and 4) were collected in mid-log phase and used to prepare membranes. Uracil permease was visualized by Western blotting. The loads were adjusted to obtain roughly equal intensities of the main permease signals from wild type and npi1 cells.



Ubiquitinated Forms of Uracil Permease Accumulate in Cells Deficient in the Internalization Step of Endocytosis

Npi1 mutant cells have reduced amounts of Ub-permease conjugates and accumulate cell-surface permease. This is consistent with the idea that ubiquitination takes place at the plasma membrane and might be required for, or even trigger, permease internalization. Ubiquitination might also occur on permease once it is internalized, and be involved in the sorting between degradative and recycling pathways. In order to choose between these two possibilities, we investigated the fate of uracil permease in mutant cells impaired in the internalization step of endocytosis.

A functional cytoskeleton is essential for the internalization step of receptor-mediated endocytosis(31, 32) . A strain carrying a conditional thermosensitive mutation in actin, act1-1, was shown to have an especially rapid and complete defect in the internalization of alpha-factor linked to its receptor, as soon as the temperature was raised to 37 °C(32) . The act1 and parental cells were transformed with multicopy plasmid encoding galactose-inducible permease. Permease activity, corresponding to plasma membrane-arrived protein, was induced at the same rate in wild type and mutant cells grown at the permissive temperature (data not shown). The turnover of uracil permease was analyzed in act1 and parental cells at both permissive and restrictive temperatures after inhibition of protein synthesis by cycloheximide. Uracil uptake was measured at various time intervals, and protein extracts were prepared and analyzed for uracil permease by immunoblots. As expected, inhibition of protein synthesis triggered the loss of uracil uptake and parallel permease degradation in wild type cells, and both events were accelerated when the temperature was raised from 24 °C to 37 °C (Fig. 5). At the restrictive temperature, and to a lesser extent at the permissive temperature, the act1 mutation provided strong protection against stress-induced loss of uracil uptake, and permease degradation. Permease, which was rapidly degraded in wild type cells at 37 °C, was maintained in act1 cells over 2 h (Fig. 5B). Thus for uracil permease, as for alpha-factor, internalization, hence subsequent degradation, were greatly impaired in act1 mutant cells at the restrictive temperature and were partially defective at the permissive temperature.


Figure 5: Defective internalization of uracil permease in act1 cells submitted to inhibition of protein synthesis. NY13 (WT) and NY279 (act1) strains transformed with the plasmid p195gF were grown at 24 °C to an A of 0.6 with galactose as carbon source. Cells were transferred or not, to 37 °C, and cycloheximide (100 µg/ml) was added to the medium 10 min later. A and B, protein extracts were prepared at the times indicated, after the addition of cycloheximide, and analyzed for uracil permease by Western immunoblotting (A, 24 °C; B, 37 °C). C, uracil uptake was measured at different times after addition of cycloheximide. Results are percent of initial activities. bullet, wild type at 24 °C; , wild type at 37 °C; circle, act1 at 24 °C; box, act1 at 37 °C.



Some Ub-permease conjugates were observed in total extracts from act1 cells grown at 24 °C (Fig. 5A, lane 5). The extent of ubiquitination was further analyzed using plasma membrane-enriched fractions from act1 and parental cells grown at 24 °C and incubated (or not) for 10 min at 37 °C with cycloheximide. Fig. 6shows an immunoblot probed with anti-(H)-ATPase and uracil permease antibodies. Whereas wild type and act1 mutant cells exhibited the same amount and pattern of the abundant, stable (H)-ATPase, they differed considerably in their pattern of permease species. Growing act1 cells contained more Ub-permease conjugates relative to the main permease signal than wild type cells (Fig. 6, lanes 1 and 2). Incubation with cycloheximide for 10 min at 37 °C led to a further enrichment in Ub-permease conjugates in act1 cells (lane 4), whereas there was a noticeable decrease in all permease specific signals in wild type cells (lane 3). Thus, both basal and stress-stimulated permease degradation involved the formation of Ub-permease conjugates at a step preceding permease internalization.


Figure 6: Ub-permease conjugates accumulate in act1 cells. NY13 (WT) and NY279 (act1) cells transformed and grown as described above were collected before (lanes 1 and 2) or after (lanes 3 and 4) incubation for 10 min at 37 °C with cycloheximide. They were used to prepare plasma membrane-enriched fractions. Aliquots containing the same amount of proteins were separated by SDS-PAGE and analyzed by immunoblotting for uracil permease and (H)-ATPase. Brackets indicate Ub-permease conjugates.



The Turnover of Uracil Permease Is Mediated by Vacuolar Proteases and Not by the Proteasome

Multi-ubiquitinated soluble conjugates are thought to be recognized and degraded by the proteasome, an essential high molecular weight proteinase lying in the cytoplasm and the nucleus of all eucaryotic cells(33) . Several short-lived proteins were indeed demonstrated to be degraded in vivo by the proteasome(21, 34) . On the other hand, plasma membrane proteins are likely to be degraded in the lysosomes/vacuoles following their endocytosis(35) . We reported previously that basal permease degradation was at least partly vacuolar, since permease accumulated in growing cells, lacking vacuolar protease activities(9) . However, there could be more than one pathway for the degradation of uracil permease. The turnover of this protein was therefore investigated in various yeast cells deficient in cytoplasmic or vacuolar proteolysis. Experiments were performed using pep4-3 mutant cells, which lack proteinase A and are therefore deficient in the activities of several other vacuolar proteinases(36) . Two types of mutants were used to check for a possible involvement of the proteasome: the double mutant pre1-1 pre2-2, impaired in the chymotrypsin-like activity of the proteasome(37) ; and cim3 and cim5 mutants, impaired in two proteasome regulatory subunits (putative ATPases)(22) .

The cim3, cim5, and pre1 pre2 mutant and isogenic wild type strains were cotransformed with multicopy plasmids encoding galactose-inducible uracil permease and Ub-proline-beta-galactosidase fusion protein (Ub-Pro-beta-gal), an artificial substrate of the Ub proteolytic pathway(25) . Exponentially growing cells were labeled for 10 min with TranS-label. Chase was then initiated by adding methionine plus cysteine, a condition that leads to a stimulated turnover of uracil permease (see ``Materials and Methods''). The half-life of uracil permease was essentially the same in each of these proteasome mutants and in the corresponding wild type cells (Fig. 7). In contrast, the short-lived Ub-Pro-beta-gal, which was rapidly degraded in wild type cells, remained almost constant during the whole chase, indicating that the proteasome function was indeed blocked in these mutant cells under our experimental conditions. A pulse-chase experiment was performed under the same conditions using pep4 and parental cells expressing galactose-inducible permease (Fig. 8). Permease was far more stable (about 5-fold) in pep4 cells than in wild type cells. These data indicate that stress-induced permease turnover depends on vacuolar proteolysis and that it is independent of the proteasome.


Figure 7: Stress-stimulated permease turnover is independent of the proteasome. A and B, WCG4a (WT) and WCG4-11/22a (pre1 pre2) cells cotransformed with pgF and p(Ub-Pro-beta-gal) were grown at 30 °C with galactose as a carbon source to mid log phase, and then incubated for 1 h at 37 °C. Cells were labeled at 37 °C with TranS-label for 5 min and chased with methionine plus cysteine for the times indicated. Samples were immunoprecipitated with anti-uracil permease and anti-beta-gal-specific antibodies. Immunoprecipitated proteins were resolved by SDS-PAGE, and the data were quantified by PhosphorImager analysis. bullet, wild type; circle, pre1 pre2. C and D, a pulse-chase analysis was performed as described in A, except that temperature was 30 °C, on YPH499 (WT, bullet), CMY763 (cim 3, circle), and CMY765 (cim5, box) cells.




Figure 8: Stress-stimulated permease turnover is dependent of vacuolar proteases. A pulse-chase experiment was performed as described in the legend of Fig. 7on W303-1B/D (WT, bullet) and IW-6A (pep4, circle) cells transformed with p195gF.



Similarly, mutations in the proteasome and the vacuolar proteolytic activities had different effects on basal permease degradation. The steady-state levels of uracil permease in the proteasome mutants were the same as in wild type cells, while these mutant cells accumulated Ub-Pro-beta-gal (data not shown). In contrast, uracil permease accumulated in exponentially growing pep4 cells(9) , while there was almost no accumulation of the (H)-ATPase (Fig. 9). We have already shown by immunofluorescence that the accumulated permease is located within structures overlapping with the vacuoles, visualized by Nomarski optics(38) . Western blot analysis of the essentially vacuolar permease from total extracts of pep4 cells gave identical patterns of permease species when immunodetection was performed with specific N-terminal or C-terminal antipeptide antibodies (Fig. 9). Therefore, uracil permease did not appear to undergo partial proteolytic processing on its way from the plasma membrane to the vacuole, an observation which confirmed that the proteasome is not involved in permease degradation. Most of the vacuole-accumulated protein appeared to be non-ubiquitinated. Although an artifactual de-ubiquitination during preparation of protein extracts cannot be entirely excluded, this observation indicated more likely that permease underwent de-ubiquitination within endocytic compartments, or after its delivery to the vacuole.


Figure 9: Uracil permease accumulates in pep4 cells as an entire protein. W303-1B/D (WT) and IW-6A (pep4) cells transformed with pfF were grown with glucose as a carbon source. Protein extracts were prepared from exponentially growing cells withdrawn at an A of 0.6. Proteins from 0.2 ml of culture were analyzed by Western blots using N-terminal permease antipeptide antibody. The nitrocellulose was then treated as described by the manufacturer (Amersham), in order to obtain the complete retrieve of the immunodetected signal, and probed simultaneously with C-terminal antipeptide antibody, and [H]-ATPase antibody.




DISCUSSION

The above data indicate that uracil permease constitutively undergoes a moderate turnover, besides stress-stimulated turnover. Both basal and stress-stimulated permease turnover involve a Npi1p-dependent ubiquitination step, which is required prior to permease endocytosis and vacuolar degradation.

Ubiquitination of uracil permease was found strikingly reduced in a npi1 mutant, deficient in the Npi1p/Rsp5p Ub-protein ligase. Despite extensive knowledge of the ubiquitin system(14) , few genes are known to encode Ub-protein ligases: the human gene encoding E6-AP (39) and the yeast UBR1 and NPI1/RSP5 genes(16, 40) . The Npi1p protein, conserved from yeast to man, has a putative phospholipid interaction motif (C2) in its N terminus, followed by several repeats of a WWP domain (41) (also referred as WW; (42) ), which has recently been implicated in mediating protein-protein interactions(43) . The C-terminal domain of Npi1p is homologous to the hect domain of the human E6-AP(4, 16, 41) . If the activity of Npi1p/Rsp5p as a Ub ligase has been documented in vitro(16) , our results provide strong evidence that Npi1p might act in vivo as the Ub ligase involved in the ubiquitination of uracil permease. Npi1p and uracil permease would then be one of the few Ub-protein ligase/substrate pairs identified so far and, to our knowledge, the first example of a pair Ub ligase/plasma membrane protein. Given the protection against ammonium-induced proteolytic breakdown of Gap1p observed in npi1 mutant cells(4) , it is highly probable that Npi1p/Rsp5p also functions as the Ub ligase required for a putative ubiquitination of Gap1p.

Little is known about the signals required for ubiquitination of cytosolic and, moreover, membrane-bound proteins. The N-end rule-based degradation signal is associated with the involvement of Ubr1 ligase, and a physiological target of this ligase has been identified in yeast(44) . The ``destruction box'' of mitotic cyclins is also a signal for ubiquitination and subsequent degradation(11) . Uracil permease has a sequence similar to the cyclin destruction box, and replacement of its invariant arginine by an alanine (R294A permease) shows that degradation of uracil permease proceeds by destruction box-dependent pathway under stress conditions (13) . In contrast, the basal turnover in growing cells might be destruction box-independent, as it is not affected in the R294A permease (data not shown). As the stability of the uracil permease depends upon the metabolic state of the cells, its ubiquitination might be regulated. There is a change in the phosphorylation of the permease in parallel with a change in turnover ( (45) and data not shown), suggesting that, as reported for a few proteins(46, 47) , the extent of phosphorylation might influence the conjugation of Ub to the permease and its subsequent instability. On the other hand, modulations of the Ub system might influence the extent of permease ubiquitination. The yeast Ub system is activated in cells faced with various adverse conditions(15) , which also lead to an increase in the permease turnover.

Two independent lines of evidence indicate that the ubiquitination of uracil permease is a cell-surface event. First, permease is stabilized at the cell-surface in npi1 mutant cells, that are deficient in permease ubiquitination. Secondly, ubiquitinated forms of uracil permease accumulate in act1 mutant cells, that are deficient in the internalization step of endocytosis. Both observations apply to basal, as well as stress-stimulated permease degradation. These data demonstrate that ubiquitination is a necessary event prior to permease internalization. Similar conclusions have been reached for three other yeast plasma membrane proteins, two ABC transporters Ste6p and Pdr5p, and Ste2p, the alpha-factor receptor, which also accumulate in ubiquitinated forms in an early endocytosis mutant(48, 49, 50) . Activation-induced multi-ubiquitination of the mammalian immunoglobulin E receptor is also a cell surface event(51) .

Multi-ubiquitinated soluble proteins are thought to be recognized and degraded by the proteasome(33, 52) . It was demonstrated recently that the proteasome is also involved in the degradation of ubiquitinated membrane-bound proteins located in the endoplasmic reticulum(53, 54) . The present results show that the half-life of the plasma membrane uracil permease is unchanged in several catalytic and regulatory mutants of the proteasome, while its degradation is strongly impaired in a pep4 mutant, deficient in vacuolar protease activities. Uracil permease does not undergo any partial proteolytic cleavage before its delivery to the vacuole since it accumulates in the vacuole of pep4 cells as an entire protein. The turnover of uracil permease therefore appears to be exclusively vacuolar, a conclusion that extends out the knowledge relative to the proteolytic breakdown of the few yeast plasma membrane proteins known to be ubiquitinated(48, 49, 55) .

The present data indicate that the ubiquitination of uracil permease functions as a signal targeting permease for internalization and subsequent degradation. Ligand-induced ubiquitination also signals Ste2p for endocytosis(49) . While ubiquitination and subsequent vacuolar degradation have been formally demonstrated for only a very few yeast plasma membrane proteins to date(48, 49, 50) , the degradation of several other transporters is known to depend on E2 or E3 enzymes(4, 56) . It is thus likely that conjugation to Ub, and subsequent endocytosis, is a general process enabling the regulated degradation of a number of plasma membrane proteins in yeast in response to various environmental cues. Whether specific machinery is involved in the recognition and internalization of ubiquitinated membrane proteins is one of the questions open for future investigation. Studying the intracellular fate of uracil permease should provide new insights into the signals, steps, and molecules involved in the yeast ubiquitin-mediated endocytic pathway.


FOOTNOTES

*
This work was supported by grants from CNRS, the University Paris7-Denis Diderot, the Fondation de la Recherche Médicale, and the Centre des Comités de Lutte contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel. 33-1-44-27-63-86; Fax: 33-1-44-27-59-94; rht{at}ccr.jussieu.fr.

(^1)
The abbreviations used are: Ub, ubiquitin; HA, hemagglutinin; Tricine, N-tris(hydroxymethyl)methylglycine; beta-gal, beta-galactosidase; PAGE, polyacrylamide gel electrophoresis.

(^2)
T. Zoladek, personal communication.


ACKNOWLEDGEMENTS

We thank Daniele Urban-Grimal for numerous discussions and comments on the manuscript, Owen Parkes for editorial assistance, and Marie-Odile Blondel for frequent help while we were doing this work. We are grateful to Marie-Renée Chevallier, Mark Hochstrasser, Carl Mann, Dieter Wolf, Barbara Winsor, D. Botstein, Christine Conesa, and Ramon Serrano for generously providing strains, plasmids, and antisera. We are indebted to Teresa Zoladek for providing information and strains prior to publication. We thank Laurence Vayssié for constructing the plasmid YEp352fF and Jean-Yves Springael for constructing the strains 27061b and 27064a.


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