A Syntaxin Homolog Encoded by VAM3 Mediates Down-regulation of a Yeast G Protein-coupled Receptor*

Christopher J. StefanDagger and Kendall J. Blumer§

From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

G protein-coupled receptors that transduce signals for many hormones, neurotransmitters, and inflammatory mediators are internalized and subsequently recycled to the plasma membrane, or down-regulated by targeting to lysosomes for degradation. Here we have characterized yeast alpha -factor receptors tagged with green fluorescent protein (Ste2-GFP) and used them to obtain mutants defective in receptor down-regulation. In wild type cells, Ste2-GFP was functional and localized to the plasma membrane and endocytic compartments. Although GFP was fused to the cytoplasmic tail of the receptor, GFP also accumulated in the lumen of the vacuole, suggesting that the receptor's extracellular and cytoplasmic domains are degraded within the vacuole lumen. Transposon mutagenesis and a visual screen were used to identify mutants displaying aberrant localization of Ste2-GFP. Mutants that accumulated Ste2-GFP in numerous intracellular vesicles carried disruptions of the VAM3/PTH1 gene, which encodes a syntaxin homolog (t-SNARE) required for homotypic vacuole membrane fusion, autophagy and fusion of biosynthetic transport vesicles with the vacuole. We provide evidence that Vam3 is required for the delivery of alpha -factor receptor-ligand complexes to the vacuole. Vam3 homologs in mammalian cells may mediate late steps in the down-regulation and lysosomal degradation pathways of various G protein-coupled receptors.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

G protein-coupled receptors (GPCRs)1 function in signal transduction pathways that allow eukaryotic cells to respond to sensory stimuli and extracellular signals, including various hormones and neurotransmitters. In response to agonist stimulation, GPCRs are regulated by phosphorylation, sequestration, and down-regulation, which limit the strength or duration of physiological responses (1, 2). Whereas down-regulation persistently attenuates signaling by targeting GPCRs for lysosomal degradation, sequestration apparently serves to reactivate desensitized receptors by allowing them to be dephosphorylated and recycled in active form to the cell surface (3, 4).

Recent studies have begun to elucidate the mechanisms governing the sequestration and down-regulation of GPCRs (2). For example, beta -arrestin acts as an adaptor that binds GPCRs and clathrin (5), recruiting receptors into coated pits that pinch off by a dynamin-dependent mechanism (6). In contrast, the molecular mechanisms governing the transport of GPCRs along subsequent steps of the endocytic pathways responsible for sequestration or down-regulation are less well understood.

GPCRs activated by peptide mating pheromones in the yeast Saccharomyces cerevisiae have provided insights into the molecular mechanisms of receptor down-regulation (7). Agonist binding triggers phosphorylation and subsequent ubiquitination of yeast alpha -factor receptors on their cytoplasmic C-terminal domains (8, 9). Ubiquitinated receptors are internalized by processes that involve clathrin, actin, myosin, and fimbrin (10), and subsequently transported through vesicular intermediates similar to early and late endosomes in mammalian cells (11, 12). In late endosomal/prevacuolar compartments, pheromone receptor trafficking is thought to converge with biosynthetic pathways that deliver precursors of resident vacuolar proteins (13-15). Subsequent events transport pheromone-receptor complexes to the vacuole where they are degraded by vacuolar hydrolases (11, 16).

Yeast pheromone receptors transit the endocytic pathway through the action of several gene products. For example, a small GTP-binding protein (Ypt5/Vps21) and an NSF homolog (Sec18), regulate fusion or maturation of early endosomes (11, 17), and two syntaxin homologs, Tlg1 and Tlg2, are required for receptor down-regulation (18).

Here we have used yeast cells expressing green fluorescent protein fused to the alpha -factor receptor (Ste2-GFP) as a new tool to localize receptors in compartments of the endocytic pathway and to identify gene products involved in receptor down-regulation. We describe evidence that a syntaxin homolog encoded by the VAM3 gene is required for delivery of Ste2 to the vacuole, a late step in the down-regulation pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials, Media, and Isotopes-- Enzymes used for recombinant DNA methods were purchased from commercial sources and used according to the suppliers' recommendations. Sources of growth media for yeast and bacterial cells have been described (19). [35S]Na2SO4 (carrier-free) was obtained from ICN. 7-Aminochloromethylcoumarin (CMAC) and monoclonal antibodies specific for carboxypeptidase Y were obtained from Molecular Probes.

Yeast Strains and Plasmids-- The S. cerevisiae strains used in these studies were JE115 (MATa ura3-52 leu2-3, 112 his3Delta -1 trp1 ste2Delta ::LEU2 sst1-Delta 5; Ref. 8), KBY49 (vam3-100 derivative of JE115), KBY62 (vam3-101 derivative of JE115), KBY58 (ste2Delta derivative of JE115), and KBY65 (vam3Delta derivative of KBY58). The unmarked ste2Delta allele of KBY58 was introduced by two-step gene deletion using JE115 and ClaI-cut YIp5ste2Delta (19), and confirmed by loss of LEU2. The VAM3 coding region was deleted using ApaI/SacI-cut pvam3::LEU2 (20), to create KBY65. To localize alpha -factor receptors in living cells, we constructed plasmid pRS314STE2-GFP, which expresses green fluorescent protein (GFP) fused to wild type receptors at their extreme C termini. Polymerase chain reaction was used to generate GFP(S65T) coding sequences flanked by BamHI restriction sites. This polymerase chain reaction product was digested with BamHI and cloned into pGEX2T (Amersham Pharmacia Biotech) that had been cut with BamHI to create pGEX2T-GFP. Plasmid pRS314STE2-GFP was created by inserting a BamHI fragment containing the GFP (S65T) coding region in the appropriate reading frame from pGEX2T-GFP into the BglII site of pRS314STE2Delta ter in which the wild type termination codon was inactivated (19). To fuse GFP to alpha -factor receptors lacking their C-terminal cytoplasmic tails, we inserted the BamHI fragment containing the GFP(S65T) coding region in the appropriate reading frame from pGEX2T-GFP into the BglII site of pRS314ste2-300ter (19), to create plasmid pRS314ste2Delta tail-GFP. To express VAM3, plasmid pRS313VAM3 was constructed by isolating a 1.8-kilobase pair XmnI-ApaI fragment encompassing the VAM3 locus from pVAM3.416 (20), and inserting it into pRS313 that had been cleaved with SmaI and ApaI. The vam3-6 temperature-sensitive allele was expressed from pvam3-6.416 (20).

Mutagenesis and Genetic Screening-- Transposon insertion mutagenesis and a visual screen were performed to identify cells that displayed aberrant localization of Ste2-GFP. Two pools of a transposon-mutagenized yeast genomic DNA library (21) cleaved with NotI were transformed into JE115 cells carrying the STE2-GFP fusion on a centromeric plasmid. Approximately 6,000 transformants were treated individually with pheromone (1 µM alpha -factor, 3 h) and screened using fluorescence microscopy for abnormal distribution of Ste2-GFP.

To identify genes affected in the mutants isolated from the visual screen, chromosomal DNA was prepared, digested with EcoRI, HindIII, or BamHI, which cleaves once in the transposable element and cleaves in the adjacent chromosomal DNA (21). Fragments containing the bla gene from the transposable element and flanking yeast genomic DNA were cloned into pCRScriptCAM SK(+) (Stratagene) cleaved with EcoRI, HindIII, or BamHI. These plasmids were introduced into Escherichia coli by selecting for resistance to ampicillin and chloramphenicol. Plasmid DNA was prepared and sequenced using a primer that anneals to the Tn3 38 base pair repeat at the left end of the transposon; this identified the chromosomal locus disrupted by the transposable element.

Pheromone Response Assays-- Quantitative bioassays (halo assays) were used to measure the apparent sensitivity of cells to pheromone-induced growth arrest (22). The relative responsiveness of cells was determined by comparing the amount of alpha -factor required to form a halo 20 mm in diameter, as indicated by interpolating dose-response curves. Quantitative mating assays were performed as described (22).

Ligand Binding and Receptor Internalization Assays-- Methods used to purify [35S]alpha -factor and perform ligand binding assays with inviable, intact cells have been described (19). Assays of cells expressing wild type or GFP-tagged receptors employed [35S]alpha -factor (30 Ci/mmol) at concentrations ranging from 1 to 10 nM. Ligand binding data were plotted according to the method of Scatchard and fitted by nonlinear least mean square regression. Nonspecific binding was determined in the presence of a 500-fold excess of unlabeled alpha -factor.

Endocytosis of the alpha -factor receptor was determined by measuring the rates that cells internalize bound 35S-labeled alpha -factor, as described previously (23). Rates of degradation of internalized 35S-labeled alpha -factor by wild type cells or vam3 mutants were determined as described previously (11, 23), with the following modifications. Cells were grown at 26 °C or 30 °C to a density of 107 cells/ml, washed, and suspended in YPB (10 mM PIPES, pH 6.0, 1 mM MgCl2, 0.1 mM EDTA, 1% yeast extract, 2% bactopeptone). 35S-Labeled alpha -factor (1 µCi, 100 nM final concentration) was bound to cells for 1 h under conditions that inhibit ligand-induced internalization of receptors (YPB at 0 °C). Unbound ligand was removed by pelleting cells, and cells were suspended in YPD medium, pH 6.0, at 26 °C, or 38 °C if vam3 temperature-sensitive mutants were analyzed. Receptor internalization was allowed to proceed. Aliquots (100 µl) were removed at various times and diluted into 10 ml 50 mM sodium citrate buffer, pH 1.1, containing 10 mM KF on ice to remove surface-bound alpha -factor. Cells were filtered, washed with 50 mM potassium phosphate buffer, pH 6.0, containing 10 mM NaN3 and 10 mM KF, and suspended in extraction buffer (23). Internalized 35S-labeled alpha -factor extracted from cells was subjected to thin layer chromatography to resolve intact and degraded alpha -factor species, as described previously (23).

Immunoblotting-- Cultures were grown to a density of 2 × 107 cells/ml in synthetic medium (SD-uracil) to select for plasmids pVAM3.416 and pvam3-6.416. Immunoblotting methods used to detect carboxypeptidase Y (CPY) in yeast whole-cell extracts were similar to those previously described (24). Cells were lysed by mechanical disruption in Laemmli sample buffer, and the protein concentration of the cleared lysates was determined by the Bradford method and adjusted to 1 mg/ml with Laemmli sample buffer prior to SDS-polyacrylamide gel electrophoresis.

Fluorescence Microscopy-- Yeast cell cultures were labeled with CMAC at a concentration of 100 µM for 15 min in synthetic medium at room temperature to examine vacuole morphology. To visualize alpha -factor receptors, cultures were grown to a density of 2 × 107 cells/ml in synthetic medium (SD-tryptophan) to select for plasmids that express GFP-tagged receptors. Cells were harvested by centrifugation, suspended in low fluorescence medium (25), and observed under an Olympus epifluorescence microscope equipped with UG-1, BP490, and BP545 dichroic filters and a cooled CCD camera (Dage).

To detect changes in the distribution of receptors upon treating cells with pheromone, we mixed MATa cells (JE115) expressing GFP-tagged receptors (from pRS314STE2-GFP) with a 4-fold excess of MATalpha cells (RK537-3B) to provide a source of alpha -factor. The cell suspension (2 µl) was placed on an agarose pad containing medium as described previously (25). Cells were imaged at room temperature on an Olympus epifluorescence microscope equipped with stage and shutter controllers. Time lapse fluorescence images were collected by acquisition of 10 focal planes (0.5 µm apart), and collapsed into a single two-dimensional image as described previously (25).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of GFP-tagged Wild Type and Truncated alpha -Factor Receptors-- To characterize the endocytic trafficking of yeast alpha -factor receptors and to identify mutants defective in this process, we needed a rapid and reliable means of examining receptor localization. Accordingly, we constructed a centromeric plasmid that expresses green fluorescent protein (GFP, S65T) fused to the extreme C-terminal cytoplasmic tail of the alpha -factor receptor, Ste2-GFP (Fig. 1). We also constructed a similar plasmid that expresses GFP fused to a truncated alpha -factor receptor lacking its cytoplasmic C-terminal tail, Ste2Delta tail-GFP, which should not be internalized from the cell surface (8, 26). These two forms of Ste2-GFP were expressed from the STE2 promoter in a ste2 null mutant and characterized with regard to signaling activity, agonist binding affinity, cell surface expression, and internalization kinetics.


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Fig. 1.   Receptor binding, expression, signaling, and internalization properties. A, agonist binding affinities and cell surface receptor expression levels of cells (JE115) expressing wild type receptors from pRS314STE2 (Ste2) or full-length GFP-tagged receptors from pRS314STE2-GFP (Ste2-GFP), and of an isogenic vam3-100 mutant (KBY49) expressing full-length GFP-tagged receptors from pRS314STE2-GFP (vam3-100). The results were obtained using two independent transformants assayed in duplicate; standard errors for Kd and Bmax values were less than 15%. B, pheromone response of cells (JE115) expressing wild type receptors from pRS314STE2 (open circles) or full-length GFP-tagged receptors from pRS314STE2-GFP (closed circles), a vam3-100 mutant (KBY49) expressing full-length GFP-tagged receptors from pRS314STE2-GFP (open squares), and a vam3-101 mutant (KBY62) expressing full-length GFP-tagged receptors from pRS314STE2-GFP (closed squares). Assays of pheromone-induced growth arrest were used. The data presented are the average of two independent experiments; standard deviations were less than 10%. C, internalization of [35S]alpha -factor by cells (JE115) expressing wild type receptors from pRS314STE2 (open circles), full-length GFP-tagged receptors from pRS314STE2-GFP (closed circles) or tail-truncated GFP-tagged receptors from pRS314ste2Delta tail-GFP (closed squares), and a vam3-100 mutant (KBY49) expressing full-length GFP-tagged receptors from pRS314STE2-GFP (open squares). Receptor internalization is indicated by the percentage of prebound [35S]alpha -factor internalized over time. Each data point represents the average of two independent assays performed in duplicate, except for cells expressing Ste2pDelta tail-GFP, which were analyzed once; standard errors are indicated.

Tagging full-length alpha -factor receptors with GFP did not appear to affect receptor signaling, agonist binding affinity, cell surface expression, or internalization. Tagged and untagged receptors displayed similar agonist binding affinities (Kd = 4.9 and 5.3 nM for Ste2-GFP and Ste2, respectively; Fig. 1A) and cell surface expression levels (Bmax = 15,000 and 19,000 sites/cell respectively, Fig. 1A). Cells expressing Ste2-GFP responded to pheromone with nearly normal efficiency (Fig. 1B) and mated as efficiently as cells expressing untagged receptors (data not shown). Furthermore, cells expressing Ste2-GFP internalized receptors at a rate indistinguishable from that of cells expressing full-length untagged receptors, as indicated by measuring rates of 35S-labeled alpha -factor uptake (Fig. 1C). Cells expressing receptors lacking their cytoplasmic C-terminal domains (Ste2Delta tail-GFP) were defective in this assay (Fig. 1C) and conferred a pheromone supersensitive phenotype identical to untagged truncated receptors (data not shown), as expected (8, 26).

Ste2-GFP Localizes to the Plasma Membrane and Putative Endocytic Compartments-- We subsequently examined the localization of full-length Ste2-GFP in the absence or presence of mating pheromone. Consistent with our previous immunofluorescence studies using fixed cells expressing myc-tagged receptors (19), Ste2-GFP was present at the cell surface (Fig. 2A), particularly in daughter cells, probably due to polarization of the secretory pathway. Ste2-GFP was also detected at the tips of cell surface projections induced 2 h after exposing cell cultures to pheromone (data not shown), similar to what has been reported using untagged receptors (27).


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Fig. 2.   Localization of GFP-tagged alpha -factor receptors. A, localization of Ste2-GFP in the absence of pheromone. Cells (JE115) expressed Ste2-GFP from pRS314STE2-GFP (top row) or Ste2Delta tail-GFP from pRS314ste2Delta tail-GFP (bottom row) to visualize receptors (GFP), and were stained with CMAC to visualize vacuoles (CMAC). Putative early endosomes (single arrowhead) and late endosomal/prevacuolar compartments (double arrowhead) are indicated. B, changes in the localization of Ste2-GFP upon pheromone stimulation. Time-lapse fluorescence video microscopy of individual cells incubated at 22 °C was used to monitor changes in the distribution of Ste2-GFP in MATa cells (JE115; indicated by asterisks in the Nomarski image in the lower right panel) in response to the pheromone secreted by neighboring MATalpha cells (RK537-3B; unlabeled in the Nomarski image and invisible in the fluorescence images because they did not express a GFP-tagged protein). At each time point, 10 z-axis fluorescence images (0.5 µm apart) were acquired and collapsed into a single image, allowing the number of endosomes containing Ste2-GFP in each cell to be counted. Over the indicated time interval, the number of early and/or late endosomes containing Ste2-GFP increased 2-4-fold.

Ste2-GFP also localized to two morphologically distinct types of intracellular vesicular structures. Smaller vesicles (usually >5/cell; Fig. 2A, single arrowhead) were present throughout the cytoplasm. The second type of vesicular structures were larger (Fig. 2A, double arrow), less numerous (typically 2-5/cell), and nearly always located next to the vacuole, which was stained with the vital dye CMAC (28).


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Fig. 3.   Localization of GFP-tagged alpha -factor receptors in wild type cells and vam3 mutants. Wild type and mutant cells expressed Ste2-GFP from pRS314STE2-GFP to visualize receptors (GFP) and were labeled with CMAC to visualize vacuolar morphology (CMAC); Nomarski images of stained cells are shown (DIC). The strains used were JE115 (wild type), KBY49 (vam3-100), and KBY62 (vam3-101). Plasmid pRS313VAM3 (CEN VAM3) expressing the wild type VAM3 gene was introduced into the indicated mutants.

Two approaches were used to determine whether the intracellular vesicles observed in cells expressing Ste2-GFP were secretory or endocytic. First, we examined the localization of Ste2Delta tail-GFP, which fails to undergo endocytosis. Ste2Delta tail-GFP was present at the cell surface, but it did not localize to intracellular vesicular structures (Fig. 2A). A small proportion of Ste2Delta tail-GFP also localized to a perinuclear ring similar to the endoplasmic reticulum, suggesting that this fusion protein has a slight biosynthetic defect. This ring structure was not the vacuole membrane because it did not stain with CMAC.

Second, because receptor internalization is stimulated by pheromone, we determined whether the number of intracellular vesicles increases when cells are exposed to the pheromone secreted by a neighboring cell of opposite mating type. For these experiments, MATa cells expressing Ste2-GFP and MATalpha cells that do not express a GFP-tagged protein were mixed and mounted on a medium-containing agarose pad. Time-lapse fluorescence video microscopy was used to follow the distribution of Ste2-GFP in single cells over time. Consistent with an endocytic origin, the number of vesicles containing Ste2-GFP increased 2-4-fold as cells responded over time to the pheromone produced by neighboring cells of opposite mating type (Fig. 2B). Increases in vesicle number were detected 20 min after cells were first exposed to pheromone, before receptor expression is induced. Results of these two experiments, and previous immunofluorescence studies of pheromone receptors (11, 14, 15), indicate that small vesicles containing Ste2-GFP are probably early endosomes, and the larger vesicles are late endosome/prevacuolar compartments.

Ste2-GFP Localizes to the Vacuole Lumen-- Although Ste2-GFP was expected to be associated with the plasma membrane, cell surface projections, and endocytic compartments, it was surprising that Ste2-GFP-derived fluorescence was also present in the lumen of the vacuole (Fig. 2A, top row). GFP accumulated in the vacuole of most cells, although the level differed significantly from cell to cell (also see Fig. 3, top row), possibly reflecting different extents of basal internalization of Ste2-GFP, rates of GFP degradation, or plasmid copy number. Localization to the vacuole lumen was confirmed by co-staining with CMAC.

Accumulation of Ste2-GFP or its degradation products in the vacuole lumen was unexpected because the GFP domain was fused to the cytoplasmic tail of the receptor, which should remain cytoplasmically disposed if endocytic trafficking to the vacuole exclusively involves fusion events between unilamellar donor and acceptor membranes. The potential significance of this observation with respect to the mechanisms of endocytic transport is presented under "Discussion."

Identification of Vam3, a Vacuolar t-SNARE That Affects the Localization of Ste2-GFP-- To identify gene products that participate in endocytic trafficking of alpha -factor receptors, we used a visual screen to isolate mutants in which the intracellular distribution of Ste2-GFP was abnormal. Transposon insertion mutagenesis was used because insertion sites are easily determined with the aid of the completed yeast genome sequence. However, this method is likely to reveal only a subset of the genes involved in endocytic trafficking because transposon insertions occur at nonrandom sites, insertions into essential genes cannot be recovered, and subtle but significant differences in receptor localization could be overlooked in a visual screen.

Mutants were identified in the following way. Pools of a transposon-mutagenized yeast DNA library were transformed into a ste2 null mutant (JE115) expressing Ste2-GFP from a centromeric plasmid; this eliminates the possibility that localization of Ste2-GFP could be affected by the presence of untagged receptors expressed from the chromosome. Transformants were screened individually in the absence and presence of pheromone for alterations in receptor distribution. Two transformants displayed a similar aberrant pattern of Ste2-GFP localization, either in the absence or presence of pheromone (data not shown). In contrast to wild type cells, these mutants displayed intense fluorescence in numerous punctate intracellular patches or vesicles (Fig. 3, second and fourth rows). Furthermore, both mutants possessed fragmented vacuoles (as revealed by CMAC staining). In contrast, when these mutants expressed endocytosis-defective receptors, Ste2Delta tail-GFP, they lacked fluorescence in intracellular vesicles (data not shown), suggesting that the localization of full-length Ste2-GFP within these vesicles requires receptor endocytosis. Thus, the mutants displayed defects in vacuolar morphology and/or targeting of endocytic vesicles containing Ste2-GFP to vacuoles.

Analysis of transposon insertion sites revealed that both mutants carried disruptions of the VAM3/PTH1 gene, which encodes a protein of 283 amino acids with similarity to yeast and mammalian syntaxins or t-SNAREs (20, 24, 29, 30). In one mutant (Fig. 3, second row) the insertion occurred at codon 112, near sequences encoding the second predicted coiled-coil domain of Vam3; this was termed the vam3-100 allele. In the second mutant (Fig. 3, fourth row) the insertion occurred at codon 268, within sequences encoding the transmembrane domain of Vam3; this was termed the vam3-101 allele. These mutations appeared to cause the mutant phenotypes because introduction of the wild type VAM3 gene on a centromeric plasmid corrected the defects in Ste2-GFP localization and vacuolar morphology (Fig. 3, third and fifth rows). Interestingly, however, the wild type VAM3 plasmid only partially corrected the phenotype of the vam3-101 mutant, because some cells in the population exhibited a wild type distribution of Ste2-GFP and intact vacuoles whereas others had a mutant phenotype (Fig. 3, fifth row). Thus, a mutation truncating Vam3 near its transmembrane domain may be partially dominant-negative. Similar truncations of other SNARE proteins are dominant-negative (31, 32).

vam3 Mutations Preserve the Signaling Activity, Expression, Ligand Binding Affinity, and Internalization of alpha -Factor Receptors-- Several observations suggested that the vam3-100 and vam3-101 mutations preserve receptor expression, signaling and internalization. First, wild type cells and vam3-100 mutants expressed receptors having similar agonist binding affinities (4.9 and 6.2 nM, respectively) and cell surface expression levels (Bmax = 14,000 and 15,000 sites/cell, respectively) (Fig. 1A). Second, vam3-100 and vam3-101 mutants expressing Ste2-GFP responded to pheromone with efficiencies similar to wild type cells expressing Ste2-GFP (Fig. 1B) and mated with normal efficiency (data not shown). Third, rates of receptor internalization were unaffected in vam3-100 mutants expressing Ste2-GFP, as revealed by measuring rates of 35S-labeled alpha -factor uptake (Fig. 1C).

Evidence That Vam3 Is Required to Deliver Receptors to the Vacuole-- Because VAM3 is dispensable for receptor expression, signaling, and internalization, it may be required specifically for the targeting and/or fusion of late endocytic vesicles with the vacuole. Consistent with this hypothesis, previous studies have shown that Vam3 localizes to the vacuole membrane where it is required for homotypic vacuole-vacuole fusion, autophagy, and targeting of biosynthetic transport vesicles that deliver carboxypeptidase Y and alkaline phosphatase to the vacuole (20, 24, 29, 30). However, the apparent mislocalization of Ste2-GFP in vam3 mutants could be due to fragmentation of the vacuole, a phenotype of vam3 mutants. Therefore, it was important to determine whether Vam3 is directly involved in the targeting or fusion of receptor-bearing endocytic vesicles to the vacuole.

Accordingly, we examined the kinetics with which untagged receptors are targeted to the vacuole by following the rate that receptor-internalized 35S-labeled alpha -factor is degraded, which requires active vacuolar hydrolases (9). Initially, these experiments were performed using wild type cells and vam3-100 and vam3-101 mutants. As shown in Fig. 4, wild type cells expressing untagged full-length receptors rapidly internalized and degraded 35S-labeled alpha -factor. After internalization was initiated, alpha -factor degradation products appeared within 30 min, and the levels of intact alpha -factor decreased with an apparent t1/2 of ~30 min, consistent with previous reports (9). In vam3-100 and vam3-101 cells (Fig. 4), both processes (loss of intact internalized alpha -factor and accumulation of alpha -factor degradation products) were essentially blocked. As expected, these defects were corrected in vam3-100 mutants bearing the wild type VAM3 gene on a centromeric plasmid (Fig. 4).


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Fig. 4.   Effects of vam3 null mutations on degradation of receptor-internalized alpha -factor. Degradation of receptor-internalized [35S]alpha -factor was followed over time as described under "Experimental Procedures." The initial time point (t = 0) was taken 10 min after internalization was initiated, before degradation occurred. Wild type receptors were expressed from pRS314STE2 in wild type cells (JE115), vam3-100 cells (KBY49), vam3-101 cells (KBY62), and vam3-100 cells (KBY49) carrying the wild type VAM3 gene on a plasmid (pRS313VAM3). A, resolution of internalized [35S]alpha -factor by thin layer chromatography and detection by autoradiography; intact and partially degraded alpha -factor are indicated. Results of a representative experiment are shown. B, quantitation of alpha -factor degradation rates. Wild type receptors were expressed from pRS314STE2 in wild type cells (JE115; open circles), vam3-100 cells (KBY49; closed circles), vam3-101 cells (KBY62; open squares), and vam3-100 cells (KBY49) carrying the wild type VAM3 gene on a plasmid (pRS313VAM3; closed squares). Data obtained from two independent experiments were analyzed by densitometry, averaged, and expressed as a percentage of intact alpha -factor present in cells at the initial time point. Standard errors are indicated.

A block in alpha -factor degradation seen in vam3 mutants could be caused by a defect in targeting of endocytic vesicles to the vacuole, or by a defect in the proteolytic processing and activation of vacuolar hydrolases required to degrade alpha -factor. To address the latter possibility, we used immunoblotting to examine the steady state levels of a mature vacuolar protease, mCPY, in wild type and vam3 mutant cells. The results indicated that the vam3-100 and vam3-101 mutations had relatively little effect on steady state levels of mCPY (Fig. 5).


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Fig. 5.   Expression levels of mature carboxypeptidase Y in wild type cells and vam3 mutants. Steady state levels of mature carboxypeptidase Y (mCPY) were determined by immunoblotting using equivalent amounts of extracts from JE115 (wild type, lane 1), KBY49 (vam3-100, lane 2), KBY62 (vam3-101, lane 3), KBY58 (wild type, lane 4), KBY65 (vam3Delta , lane 5), KBY65 carrying the vam3-6 allele on plasmid pvam3-6.416 (vam3-6, lane 6), and KBY65 carrying the wild type VAM3 gene on plasmid pVAM3.416 (VAM3, lane 7). Monoclonal antibodies specific for CPY and an enhanced chemiluminescence detection system were used.

As an alternative means of determining whether defects in the degradation of receptor-internalized alpha -factor are due directly to a targeting defect, we analyzed a vam3 temperature sensitive mutant (vam3-6) (20). This allele of VAM3 produces a protein product that is rapidly inactivated (within 10 min) upon temperature shift. Therefore, growth of cells at the permissive temperature (26 °C) should allow vacuolar hydrolases to accumulate to normal levels, and, after a brief shift to the nonpermissive temperature (38 °C), alpha -factor uptake and degradation assays can be performed. However, this requires that the levels of mature CPY are unaffected after a brief incubation (15 min) of vam3-6 mutants at the nonpermissive temperature (38 °C). This prerequisite was fulfilled, as revealed by immunoblotting experiments (Fig. 5).

The rates of 35S-labeled alpha -factor degradation in wild type and vam3-6 mutant cells were measured following a 15-min shift to the nonpermissive temperature. As shown in Fig. 6, wild type cells expressing untagged full-length receptors internalized and degraded 35S-labeled alpha -factor at 38 °C. Degradation products began to accumulate 30 min after internalization was initiated, and increased over time as intact alpha -factor disappeared (Fig. 6). The kinetics of alpha -factor degradation in wild type cells shifted to 38 °C were somewhat slower (t1/2 of ~50 min) than in previous experiments, probably because the actin cytoskeleton, which is required for receptor internalization, is depolarized transiently at elevated temperatures (33). Nevertheless, at the nonpermissive temperature, alpha -factor degradation was ~3-fold slower in the vam3-6 mutant (t1/2 >90 min) than in wild type cells, and a more severe defect was observed in vam3Delta mutants (Fig. 6). The residual degradation of alpha -factor that occurs in the vam3-6 mutant is probably carried out by partially processed or misprocessed peptidases that accumulate in prevacuolar compartments at the nonpermissive temperature (20). As expected, these defects in alpha -factor degradation were corrected by introducing the wild type VAM3 gene on a centromeric plasmid (Fig. 6). Receptor degradation is probably also blocked in vam3 mutants because previous investigations have indicated that alpha -factor and its receptor transit the same pathway to the vacuole (7, 34). Therefore, the results of our study suggest that Vam3 has a direct role in the down-regulation pathway by targeting or fusing receptor-bearing endocytic vesicles with the vacuole.


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Fig. 6.   Effects of a vam3 temperature-sensitive mutation on degradation of receptor-internalized alpha -factor. A, resolution of internalized [35S]alpha -factor by thin layer chromatography and detection by autoradiography; intact and partially degraded alpha -factor are indicated. Wild type receptors were expressed from pRS314STE2 in wild type cells (JE115; wild type), and in a vam3Delta mutant (KBY65) carrying an empty vector (pRS416; vam3Delta  + vector), a plasmid expressing the vam3-6 temperature-sensitive allele (pvam3-6.416; vam3Delta  + CENvam3-6), or a plasmid expressing the wild type VAM3 gene (pVAM3.416; vam3Delta  + CENVAM3). Results of a representative experiment are shown. B, quantitation of alpha -factor degradation rates. Wild type receptors were expressed from pRS314STE2 in wild type cells (JE115; open circles), and in a vam3Delta mutant (KBY65) carrying an empty vector (pRS416; closed circles), a plasmid expressing the vam3-6 temperature-sensitive allele (pvam3-6.416; open squares) or a plasmid expressing the wild type VAM3 gene (pVAM3.416; closed squares). Data obtained from two independent experiments were analyzed by densitometry, averaged, and expressed as a percentage of intact alpha -factor present in cells at the initial time point. Standard errors are indicated.


    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Ste2-GFP as a Marker of Endocytic Trafficking in Living Cells-- To study G protein-coupled receptor trafficking and down-regulation in yeast, we have utilized cells that express green fluorescent protein fused to the alpha -factor receptor, Ste2-GFP. Similar to studies of mammalian GPCRs tagged with GFP (35, 36), we find that Ste2-GFP exhibits essentially normal agonist binding affinity, cell surface expression, and internalization rates. Moreover, the distribution of Ste2-GFP at the plasma membrane in naive or pheromone-treated cells is similar to that of untagged or myc-tagged receptors (19, 27). Ste2-GFP also localizes to endosomes and prevacuolar compartments, which upon pheromone stimulation increases in accord with faster rates of receptor internalization (37).

Surprisingly, cells expressing Ste2-GFP accumulate GFP in the vacuole lumen, even though GFP has been fused to the cytoplasmic C-terminal tail of the receptor. Lumenal accumulation of GFP requires receptor internalization because GFP fluorescence is not detected in the vacuole lumen when cells express an endocytosis-defective form of Ste2-GFP, or GFP alone (38).

Although the mechanisms responsible for lumenal accumulation of GFP remain to be determined, we suggest two possibilities that would allow both the extracellular and cytoplasmic domains of the receptor to gain access to the lumen of the vacuole (Fig. 7). In one model, endocytic transport vesicles bearing Ste2-GFP are engulfed by an acceptor compartment, such as the vacuole. Alternatively, the membranes of late endosomes/prevacuoles or vacuoles bearing Ste2-GFP could involute and pinch off into the lumen. This is consistent with previous studies showing that Ste2 and internalized gold particles are present in multivesicular bodies (11, 12). An engulfment or involution mechanism may be an important step in the receptor down-regulation pathway because either would provide a means of degrading bound alpha -factor and the extracellular and intracellular domains of the receptor.


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Fig. 7.   Hypothetical mechanisms whereby GFP derived from Ste2-GFP accumulates in the vacuole lumen. GFP-tagged alpha -factor receptors (Ste2-GFP), and various compartments in the endocytic pathway are indicated. See "Discussion" for details.

Role of Vam3, a Vacuolar t-SNARE Homolog, in Receptor Down-regulation-- By using a visual screen to identify mutants defective in the localization of Ste2-GFP, we obtained recessive and partially dominant mutations in the VAM3 gene. VAM3 was first identified in a screen for mutants displaying fragmented vacuoles (39) and subsequently has been shown to encode a vacuole membrane-localized syntaxin (t-SNARE) homolog required for three processes: docking and fusion of autophagic and biosynthetic transport vesicles to the vacuole (20, 24, 30, 40), and homotypic fusion of vacuolar membranes in vitro (29).

Our studies suggest that Vam3 is required for endocytic trafficking of alpha -factor receptors to the vacuole. We find that in vam3 null mutants or vam3-6 temperature-sensitive mutants shifted briefly to the nonpermissive temperature, the proteolytic degradation of alpha -factor internalized by receptors is strongly inhibited. Because vam3 mutants do not have significant deficits in the levels of mature hydrolases within the vacuole, we suggest that Vam3 has a direct role in the targeting of vesicles bearing receptor-ligand complexes to the vacuole. Furthermore, because Vam3 is required for biosynthetic transport of vacuolar hydrolase precursors, our findings reinforce the current view that endocytic trafficking of receptor-ligand complexes converges at a prevacuolar step with pathways that deliver immature soluble hydrolases to the vacuole.

Although vam3 null mutants are defective in a late step in the receptor down-regulation pathway, they appear to be normal with respect to pheromone response, receptor polarization to the tips of cell surface projections and mating. This is expected because receptor internalization is unaffected in vam3 mutants. Thus, even in vam3 mutants receptors are effectively internalized and sequestered from the cell surface.

Whereas yeast mating pheromone receptors are down-regulated upon agonist exposure, mammalian GPCRs can be internalized and subsequently recycled to the plasma membrane, or down-regulated and degraded. For example, beta 2-adrenergic receptors can be internalized and recycled in response to short term agonist stimulation, whereas thrombin receptors are down-regulated (41). Understanding how these different fates are achieved requires determining which endocytic compartments are used by both the recycling and down-regulation pathways, and which compartments are used uniquely by each pathway. As suggested by our analysis of vam3 mutants and studies of other SNARE mutants (31, 32), it may be possible to define the compartments transited by recycled versus down-regulated GPCRs in mammalian cells by using dominant-negative forms of various syntaxins to block endocytic transport at specific steps. We anticipate that homologs of Vam3 in mammalian cells will be required for the down-regulation and degradation of various GPCRs.

    ACKNOWLEDGEMENTS

We thank T. Darsow and S. Emr for providing strains and plasmids, M. Goebl for the transposon insertion library, M. Overton for constructing KBY58, and M. Linder and members of our laboratory for comments on the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant GM44592 (to K. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Division of Cellular & Molecular Medicine, Howard Hughes Medical Institute, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0668.

§ Established Investigator of the American Heart Association. To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-1668; Fax: 314-362-7463; E-mail: kblumer{at}cellbio.wustl.edu.

The abbreviations used are: GPCR, G protein-coupled receptor; CMAC, 7-aminochloromethylcoumarin; CPY, carboxypeptidase Y; G protein, guanine nucleotide-binding regulatory protein; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; SNARE, soluble NSF attachment protein receptor.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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