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Vol. 17, Issue 7, 3031-3050, July 2006

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Amino Acids Regulate Retrieval of the Yeast General Amino Acid Permease from the Vacuolar Targeting Pathway

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Submitted July 25, 2005; Revised April 10, 2006; Accepted April 14, 2006
Monitoring Editor: Howard Riezman

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Intracellular sorting of the general amino acid permease (Gap1p) in Saccharomyces cerevisiae depends on availability of amino acids such that at low amino acid concentrations Gap1p is sorted to the plasma membrane, whereas at high concentrations Gap1p is sorted to the vacuole. In a genome-wide screen for mutations that affect Gap1p sorting we identified deletions in a subset of components of the ESCRT (endosomal sorting complex required for transport) complex, which is required for formation of the multivesicular endosome (MVE). Gap1p-GFP is delivered to the vacuolar interior by the MVE pathway in wild-type cells, but when formation of the MVE is blocked by mutation, Gap1p-GFP efficiently cycles from this compartment to the plasma membrane, resulting in unusually high permease activity at the cell surface. Importantly, cycling of Gap1p-GFP to the plasma membrane is blocked by high amino acid concentrations, defining recycling from the endosome as a major step in Gap1p trafficking under physiological control. Mutations in LST4 and LST7 genes, previously identified for their role in Gap1p sorting, similarly block MVE to plasma membrane trafficking of Gap1p. However, mutations in other recycling complexes such as the retromer had no significant effect on the intracellular sorting of Gap1p, suggesting that Gap1p follows a genetically distinct pathway for recycling. We previously found that Gap1p sorting from the Golgi to the endosome requires ubiquitination of Gap1p by an Rsp5p ubiquitin ligase complex, but amino acid abundance does not appear to significantly alter the accumulation of polyubiquitinated Gap1p. Thus the role of ubiquitination appears to be a signal for delivery of Gap1p to the MVE, whereas amino acid abundance appears to control the cycling of Gap1p from the MVE to the plasma membrane.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
The family of amino acid permeases expressed in Saccharomyces cerevisiae fall into two different classes with respect to their regulation. Most of the permeases are expressed constitutively and import specific amino acids or chemically related amino acids. A second class of permeases are most highly expressed under conditions of nitrogen limitation and are thought to scavenge amino acids for their use as a source of nitrogen (Magasanik and Kaiser, 2002). The major nitrogen-scavenging permeases are Gap1p, which transports all naturally occurring amino acids with a high capacity (Grenson et al., 1970; Jauniaux and Grenson, 1990), and Put4p, which is specific for proline (Vandenbol et al., 1989). In part, the activity of Gap1p and Put4p is determined by an intracellular sorting decision, which depends on the nitrogen source in the growth medium. When yeast cells are grown on medium that lacks amino acids, Gap1p and Put4p are delivered to the plasma membrane where they are active for amino acid uptake. However, when cells are grown on a medium rich in an amino acid such as glutamate, Gap1p and Put4p are sorted to the vacuole for degradation (Roberg et al., 1997a; Chen and Kaiser, 2002).

Mutations that affect the intracellular sorting of Gap1p can be classified into two general types. The first type includes mutations that cause constitutive sorting of high levels of Gap1p to the plasma membrane. This category includes in the genes RSP5, BUL1, BUL2, and DOA4, all of which either partially or completely block the intracellular ubiquitination of Gap1p, which serves as a tag for sorting to the vacuole (Helliwell et al., 2001; Soetens et al., 2001; Springael et al., 1999). Ubiquitination constitutes a common signal for endocytic internalization of a variety of plasma membrane proteins (reviewed by Hicke, 1997; Horák, 2003). The second category includes mutations that cause constitutive sorting of Gap1p to the vacuole, regardless of the nitrogen source in the growth medium. Among the genes belonging to this category are the genes LST4, LST7, and LST8, which were initially identified because of their lethality in combination with a thermosensitive allele of SEC13 (Roberg et al., 1997b). LST8 encodes a positively acting component of the TOR pathway that affects Gap1p sorting by negatively regulating the transcription factors Rtg1/3p and Gln3p, thereby limiting the synthesis of -ketoglutarate, glutamate, and glutamine (Chen and Kaiser, 2003). The roles of LST4 and LST7 have not been elucidated. A fundamental relationship between mutations of the two types is that, when a mutation that blocks ubiquitination and causes constitutive sorting to the plasma membrane is combined with a mutation that causes constitutive sorting to the vacuole, the double mutants invariably show constitutive sorting to the plasma membrane. This finding has led to the hypothesis that ubiquitination of Gap1p precedes sorting of Gap1p to a compartment in which the mutations of the second type can exert their effect (Helliwell et al., 2001).

A variety of studies have revealed that sorting of most plasma membrane proteins for degradation in the vacuolar lumen occurs through the maturation of the late endosome or prevacuolar compartment into multivesicular endosomes (MVEs; reviewed by Katzmann et al., 2002; Raiborg et al., 2003; Babst, 2005). The protein machinery required for MVE formation was discovered by identification of class E vps mutants. These mutants share a common phenotype characterized by accumulation of proteins destined for the vacuole in an enlarged prevacuolar (class E) compartment (Raymond et al., 1992). Most of the class E VPS (vacuolar protein sorting) genes encode components of three protein complexes (endosomal sorting complex required for transport [ESCRT]), designated ESCRTI, ESCRTII, and ESCRTIII, which are required for the formation of inwardly budding luminal vesicles that fill the interior of MVEs. The luminal vesicles typically contain membrane proteins that arrive in the prevacuolar compartment either by vesicular trafficking from the Golgi or by endocytosis and are eventually degraded completely in the vacuole lumen. Mutations in ESCRT complex prevent formation of inwardly budding vesicles, leading to formation of a class E compartment rather than a MVE. Impaired formation of inwardly budding vesicles can block recycling of proteins such as Vps10p from the prevacuolar compartment to the Golgi, thus leading to their accumulation in the resulting class E compartment (Babst et al., 1998, 2000, 2002a, 2002b; Babst, 2005; Katzmann et al., 2001, 2003; Bilodeau et al., 2003; Odorizzi et al., 2003; Luhtala and Odorizzi, 2004).

Most membrane proteins that are normally delivered to the lumen of the vacuole require modification by ubiquitination as a signal for being packaged into luminal vesicles of the MVE. Recent work has demonstrated that in many cases Rsp5p is directly required at the MVEs for modification and adequate sorting of membrane proteins (Blondel et al., 2004; Dunn et al., 2004; Katzmann et al., 2004; Morvan et al., 2004). Instead of causing an increase in plasma membrane sorting, as observed for Gap1p (Helliwell et al., 2001), in these cases an rsp5 mutation causes the cargo to accumulate in the delimiting membranes of the endosomal and vacuolar compartments.

Deubiquitination of the MVE cargo before its internalization into luminal vesicles is also important for the proper sorting of MVE cargo proteins. For example, The ubiquitin (Ub) C-terminal hydrolase encoded by DOA4 plays a major role at this step in the deubiquitination of different MVE cargoes. A doa4 mutation causes mistargeting of MVE cargo proteins and a failure to recycle ubiquitin, which results in depletion of intracellular pools of free ubiquitin (Swaminathan et al., 1999; Amerik et al., 2000; Dupré and Haguenauer-Tsapis, 2001).

Although defects in cargo ubiquitination, recognition by ESCRT machinery, and deubiquitination at the MVEs may result in the accumulation of proteins in endosomal and perivacuolar membranes, recent studies indicate the existence of alternative pathways that allow recycling of plasma membrane proteins from the latest stages of lysosomal/vacuolar sorting (Babst et al., 2000; Nikko et al., 2003; Bugnicourt et al., 2004; Pizzirusso and Chang, 2004).

Here we present the results of a genome-wide screen used to identify new functions involved in the control of Gap1p intracellular sorting. We find that mutations in all of the class E VPS genes involved in the MVE pathway cause missorting of intracellular Gap1p to the plasma membrane. Our results show both that Gap1p must follow the MVE pathway in order to be delivered to the vacuole and that a deficiency in the MVE machinery allows efficient retrieval of Gap1p from this intracellular compartment (probably via the trans-Golgi) to the plasma membrane rather than causing its accumulation in the class E compartment. Evaluation of the Gap1p sorting in cells grown on different nitrogen sources shows that cycling of Gap1p from the MVE to the plasma membrane is the main step in the intracellular trafficking itinerary of Gap1p that is regulated by amino acids.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Strains, Plasmids, and Media
A genome-wide screen was performed using the collection of kanMX-marked deletion mutants in nonessential genes of S. cerevisiae, from EUROSCARF (http://www.uni-frankfurt.de/fb15/mikro/euroscarf/data/by.html; Brachmann et al., 1998). All the null mutants utilized in our screen are in the BY4741 (Y00000) and BY4742 (Y10000 [GenBank] ) genetic backgrounds (MATa his31 leu22 met150 ura30 and MAT his31 leu22 lys20 ura30, respectively). Characteristically, strains of this genetic background (derived from S288C) produce high Gap1p and Put4p activity when ammonia is used as nitrogen source (Courchesne and Magasanik, 1983). All of the mutants assayed for Gap1p activity and localization were reconstructed in our laboratory genetic background, also derived from S288C, and are listed in Table 1. All complete gene deletions described here were obtained by replacement of the functional ORF of the corresponding gene by homologous recombination with either a kanMX4/6 or a natMX4 cassette (Longtine et al., 1998; Goldstein and McCusker, 1999) in the wild-type strain CKY835.

Assays for Amino Acid Uptake and -Galactosidase
Assays of the rate of uptake of radiolabeled amino acids were performed as described by Roberg et al. (1997b). -Galactosidase activity was measured using the permeabilized cell method (Adams et al., 1996).

Screen for Mutations That Affect Gap1p Activity
A primary screen was performed using master 96-well microtiter dishes containing the entire collection of deletions in nonessential genes of the EUROSCARF collection. Approximately 4859 BY strains of each haploid mating type were grown on solid YPD medium overnight and transferred to 96-well plates. Strains were spotted onto plates of minimal urea medium supplemented with amino acids and containing five different concentrations (0, 8, 30, 60, 100 mg/l) of ADCB. Sensitivity or resistance to ADCB was scored after growth for 3–4 d. As controls on each screening plate, we used the wild-type standard strain, BY4741 (Y00000) and the mutant strains lst4 (Y05026), which causes reduced levels of Gap1p activity (Roberg et al., 1997b), and gln3 (Y00173), which causes increased levels of Gap1p activity (Stanbrough and Magasanik, 1995; Chen and Kaiser, 2002). Candidate strains with either increased sensitivity or resistance to ADCB were reconfirmed in both mating types (BY4741 and BY4742 backgrounds). Subsequent to the initial screening of the deletion strains, Gap1p activity and transcription were determined after transformation of strains with pCK283 to render the strains prototrophic (so that amino acid supplements would not be necessary in the growth medium) and with pMS29 as a reporter to monitor GAP1 expression.

Fluorescence Microscopy
Labeling with FM4-64 (Molecular Probes, Eugene, OR) was performed according to the procedure described in Vida and Emr (1995). For GFP localization studies, Gap1p-GFP–expressing cells were grown to exponential phase in ammonia liquid cultures. Glutamate was added to a final concentration of 3 mM and the cells were incubated for 30 min to 1 h at 24°C. Cells were then collected by centrifugation, washed, and suspended in 1 M Tris, pH 8.0, 5% NaN₃ as described by Urbanowski and Piper (1999). This treatment stops the membrane traffic and provides alkaline conditions optimal for GFP fluorescence imaging. Images were captured with a Nikon E800 microscope (Melville, NY) equipped with a Hamamatsu digital camera (Bridgewater, NJ). The FITC filter set was used for imaging of FM4-64 detection and the chroma filter set at 41012 was used for GFP detection. Improvision OpenLabs 2.0 software (Lexington, MA) was used to process images.

FM4-64 Recycling Assay
To measure recycling of endocytosed membranes back to the cell surface, we carried out FM4–64 recycling assays as described in Wiederkehr et al. (2000). Fluorescence was recorded using a Fluorolog spectrofluorometer (Jobin/Yvon, Horiba, Irvine, CA).

Carboxy peptidase Y Secretion Assay
Detection of secreted carboxy peptidase Y (CPY) was carried out as described by Lafourcade et al. (2004).

Immunoprecipitation and Immunoblotting of Ubiquitin Conjugates of Gap1p
For the detection of Gap1 protein levels, two OD₆₀₀ of cells were collected at the required times after shifting nitrogen sources and protein extracts carried out by following a protocol from Adams et al. (1996). Proteins were resolved by 8% SDS-PAGE and detected by immunoblot using rabbit polyclonal anti-Gap1p antibody (made as explained in Risinger and Kaiser, unpublished results) at 1:2000 dilution, mouse monoclonal anti-3-phosphoglycerate kinase (1:1000, Molecular Probes) used for simultaneous detection of Pgk1p as a loading control, and HRP-conjugated donkey anti-rabbit IgG or HRP-coupled sheep anti-mouse IgG (1:10,000 dilution, Amersham, Indianapolis, IN).

For the detection of Gap1p-HA and its ubiquitin conjugates, Gap1p was immunoprecipitated and then detected by immunoblotting following an adaptation of the protocol described by Laney and Hochstrasser (2002). Strains were transformed with pP_CUP1-myc-UBI (pCK322) and then cultured in minimal urea medium to an initial OD₆₀₀ of 0.01/ml. myc-Ubi expression was induced for 16 h with 1 µM CuSO₄. An equivalent to 10 OD₆₀₀ units of cells were collected on 0.45-µm nitrocellulose filters once cultures reached 0.4 OD₆₀₀/ml. Cells were washed twice in 10 mM NaN₃, suspended in 200 µl of SDS buffer containing NEM and protease inhibitors (1% (wt/vol) SDS; 45 mM Na-HEPES, pH 7.5; 50 mM NEM; 1 mM phenylmethylsulfonyl fluoride (PMSF); 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin), and lysed with glass beads by vortexing 5 min at room temperature. Lysates were diluted in 700 µl of Triton lysis buffer with NEM and protease inhibitors (1% [vol/vol] Triton X-100; 150 mM NaCl; 50 mM Na-HEPES, pH 7.5; 5 mM Na-EDTA; 10 mM NEM; 1 mM PMSF; 0.5 µg/ml leupeptin; 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin), and any remaining cell debris was removed by centrifugation at 4°C, 14,000 x g. Samples were preadsorbed for 1 h at room temperature with 40 µl of a 20% suspension of protein G-Sepharose 4 fast flow (Amersham Pharmacia Biotech, Piscataway, NJ) followed by brief centrifugation to remove the beads. This procedure was repeated twice. Immunoprecipitation was carried out by overnight incubation at 4°C with 10 µl of monoclonal antibody preparation (rat anti-HA [3F10]; Roche, Indianapolis, IN), followed by overnight incubation at 4°C with 60 µl of protein G-Sepharose suspension. The beads were washed five times with 1% (vol/vol) Triton in phosphate-buffered saline buffer containing NEM 10 mM, 1 mM PMSF, 0.5 µg/ml leupeptin, 2 µg/ml aprotinin, and 0.7 µg/ml pepstatin. Immunoprecipitates were solubilized by incubation in sample buffer for 1 h at 37°C and resolved by 8% SDS-PAGE. Antibodies used for immunoblotting included mouse anti-HA monoclonal 16B12 (BAbCO, Richmond, CA) at 1: 1000 dilution; mouse anti-myc, monoclonal 9E10 (Santa Cruz Biotechnology, Santa Cruz, CA) at 1: 500 dilution; and HRP-coupled sheep anti-mouse serum at 1: 10,000 dilution (Amersham Pharmacia).

Pulse-Chase Experiments
To assess the inhibition of bulk translation caused by cycloheximide, cells growing exponentially in minimal glutamate medium lacking methionine were suspended in fresh medium at 5 OD₆₀₀/ml and pulse-labeled for 4 min by addition of 30 µCi of [³⁵S]methionine and [³⁵S]cysteine (EXPRESS, NEN, Boston, MA) per OD₆₀₀. Cycloheximide was then added and cells were incubated for 30 min at room temperature. Metabolic labeling of proteins was stopped by the addition of unlabeled 10 mM methionine and 10 mM cysteine before washing twice with ice-cold 20 mM NaN₃. A 0.4-ml aliquot of culture was suspended in 200 µl of sample buffer containing protease inhibitors as above. Cells were lysed by vortexing with glass beads for 5 min at 4°C, and boiled for 1 min, 20-µl samples were resolved by SDS-PAGE (8% gel), and labeled proteins were detected with a 445si PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

RESULTS

Identification of VPS Genes Involved in the Sorting of Gap1p
To identify new genes that govern proper sorting of nitrogen-regulated permeases, we screened 4859 haploid deletion mutants in nonessential genes of S. cerevisiae from the EUROSCARF collection. For the first step of selection in this screen we used the toxic proline analog ADCB, which is taken up primarily by Gap1p and Put4p permeases in cells grown on ammonia or urea as a nitrogen source (Roberg et al., 1997a). Mutations causing increased Gap1p and Put4p activities display enhanced sensitivity to ADCB, whereas mutations that reduce these activities are more resistant to this compound (Roberg et al., 1997b). By spotting each strain from the collection onto plates with minimal urea medium containing different concentrations of ADCB, a total of 162 mutants showing sensitivity to a normally sub-LC (<12.5 mg/l) of ADCB were identified. (An additional 118 mutants that displayed resistance to ADCB were identified, and these mutants will be described elsewhere.)

Enhanced sensitivity to ADCB, indicating increased activity of Gap1p and Put4p could be due to an effect on either the intracellular sorting of nitrogen-regulated permeases or their level of expression. To distinguish these possibilities, we assayed the effect of each mutant on Gap1p activity and expression. Gap1p activity was measured by the rate of uptake of [¹⁴C]citrulline, which is transported only by Gap1p. For these experiments, uptake of [¹⁴C]arginine was used to control for nonspecific effects on permease activity. GAP1 transcription was evaluated by measuring the -galactosidase activity expressed from a P_GAP1-lacZ reporter. These assays showed that 73 of the mutants had significantly increased Gap1p activity, and that for 70 of these mutants the increase in Gap1p activity was not due to an increase in GAP1 transcription (our unpublished data). Among the mutants that displayed increased Gap1p activity but normal GAP1 transcription, we almost the entire set of class E of VPS, which code for members of the ESCRT machinery acting at the MVE (Babst, 2005). The phenotypes obtained in the ADCB screen for mutations in all known VPS genes are summarized in the Table 3.

did4, vps4, vps27,

doa4

bul1 bul2

View larger version (21K): [in this window] [in a new window]   Figure 1. Null mutations in class E vps genes cause increased activity of Gap1p. (A) Wild-type (CKY835), lst4 (CKY695), bul1 bul2 (CKY698), did4 (CKY839), vps4 (CKY836), vps27 (CKY837), and doa4 (CKY838) strains were spotted as serial dilutions onto minimal urea medium, with or without 7 mg/l of ADCB. (B) The same strains were grown in liquid minimal urea medium and assayed for [14C]citrulline and [14C]arginine uptake. (C) Wild-type (CKY835) and strains carrying a genomic PADH1-GAP1 replacement of the endogenous GAP1 gene (CKY833), combined with did4 (CKY840), vps4 (CKY841), vps27 (CKY842), and doa4 (CKY843) mutations, were assayed for [14C]citrulline and [14C]arginine uptake. The data are expressed as a percentage of the rate of uptake for the wild-type strain and represent the mean for at least four independent experiments. Error bars, 1 SD.

Membrane proteins targeted to the vacuole through the MVE pathway are sorted into the membrane of inwardly budding vesicles generated at the MVE, which are ultimately delivered into the vacuolar lumen when the MVE fuses with the vacuole (Reggiori and Pelham, 2001; Katzmann et al., 2002; Babst, 2005). Accordingly, Gap1p-GFP in a wild-type strain growing in a high concentration of the amino acid glutamate, which induces Gap1p vacuolar sorting, localizes within the vacuolar lumen (Figure 2).

View larger version (44K): [in this window] [in a new window]   Figure 2. Gap1p is sorted into the vacuolar lumen. A wild-type strain expressing a genomic GFP-tagged version of GAP1 (CKY834) was grown to exponential phase in minimal ammonia medium. Glutamate was added to a final concentration of 3 mM, and cells were incubated for an additional hour. Cells were then labeled with the vacuolar membrane staining dye FM4-64 for 30 min, followed by a 60-min chase before microscopy. Cells were imaged using a UV fluorescence microscope with a GFP filter (GFP), a rhodamine filter (FM4-64), and by Nomarski optics (DIC).

Gap1p Sorting Responds to Amino Acids in MVE Mutants
We asked whether the increased levels of Gap1p activity in class E vps mutants might be due to a defect in responding to amino acids. Mutations in MKS1 display high, unregulated expression of TCA pathway enzymes responsible for -ketoglutarate formation and thus produce high levels of glutamate and glutamine (Butow and Avadhani, 2004). We have found that high amino acid content of mks1 mutants cause Gap1p to be sorted to the vacuole (Chen and Kaiser, 2002). Mutants defective in Gap1p polyubiquitination, when combined with mks1, showed highly increased levels of active Gap1p localized to the cell surface (as observed in Figure 3, A and B, for the triple mutant mks1 bul1 bul2). This result establishes that if Gap1p cannot be ubiquitinated, that Gap1p sorting no longer responds to high levels of amino acids. By contrast, a double mutant mks1 vps27 exhibited low levels of Gap1p activity (Figure 3A). Examination of Gap1p-GFP in mks1 vps27 strains revealed that Gap1p-GFP accumulated in endosomal/perivacuolar membranes, suggesting that Gap1p rerouting to the vacuolar sorting pathway in response to high internal amino acid concentrations is not impaired in this double mutant (Figure 3B).

View larger version (26K): [in this window] [in a new window]   Figure 3. Inactivation of Gap1p activity by amino acids occurs in the class E vps mutants but not in mutants impaired for Gap1p ubiquitination. (A) [14C]citrulline and [14C]arginine uptake as assayed in a mutant with increased levels of catabolic amino acids, mks1 (CKY758), combined with the mutant background defective in Gap1p polyubiquitination, bul1 bul2 (mks1 bul1 bul2; CKY923) or the mutant class E background, vps27 (mks1 vps27; CKY924). Activities for the wild-type strain (CKY835) and the mutant controls bul1 bul2 (CKY698) and vps27 (CKY837) grown in parallel are provided for comparison. The cell cultures were grown in minimal ammonia medium. Averaged data are expressed as in Figure 1. (B) Gap1p-GFP localization is shown in cells from the mutants mks1 (CKY867), mks1 bul1 bul2 (CKY926), and mks1 vps27 (CKY927), grown in minimal medium with ammonia as a nitrogen source. Gap1p-GFP images of the wild-type strain (CKY834), the mutant controls bul1 bul2 (CKY925), and vps27 (CKY851), grown identically, are shown for comparison. (C) A [14C]citrulline uptake time course from the following strains: wild-type (CKY835; ), end3 (CKY934; ), did4 (CKY839; ), vps4 (CKY836; ), and vps27 (CKY837; ), growing in liquid minimal ammonia medium (left panel) or ammonia medium plus 3 mM glutamate, added when the strains were at OD600 of 0.2/ml (right panel), is shown. These graphics show the averaged data between three independent experiments.

vps4, vps27,

did4

end3

These data indicate that, although class E vps mutants caused increased traffic of Gap1p to the plasma membrane, these mutants can respond normally to high external and internal amino acid concentrations by redirecting Gap1p away from the plasma membrane to the vacuolar targeting pathway. This behavior clearly distinguishes class E vps mutants from mutations that affect Gap1p ubiquitination, such as a bul1 bul2 double mutant, because the latter not only cause an increase in Gap1p activity, but also render Gap1p sorting insensitive to the effect of amino acids.

Gap1p Traffic to the Plasma Membrane in a Class E vps Mutant Is Blocked by an LST4 Mutation
Mutations in the gene LST4 dramatically reduce the activity of Gap1p because they cause constitutive sorting of Gap1p to the vacuole, regardless of the nitrogen source in the growth medium (Roberg et al., 1997b). Nevertheless, the lst4 phenotype can be completely suppressed by a bul1 bul2 double mutant, suggesting that Gap1p encounters the sorting step specified by Bul1p and Bul2p before the step that depends on Lst4p (Helliwell et al., 2001). In contrast to the behavior shown by the triple mutant bul1 bul2 lst4, a simultaneous mutation of the gene LST4 completely rescued growth of the bro1, vps4, vps27, and did4 mutants on plates of minimal ammonia (Figure 4A) or urea (our unpublished data) medium containing 7 mg/l of ADCB. Uptake assays to measure Gap1p activity levels were consistent with the sensitivity to ADCB. Double mutants, lst4 vps4, lst4 vps27, and lst4 did4, all had 5% of the Gap1p activity as the corresponding single class E mutant (Figure 4B). Null double mutants with lst4 for the remaining class E genes showed similar reductions of Gap1p activity except for an lst4 bro1 double mutant, which exhibited 20% of the activity as a bro1 single mutant.

View larger version (20K): [in this window] [in a new window]   Figure 4. The lst4 mutation has a similar effect on Gap1p trafficking as glutamate and high amino acid concentrations. The strains doa4 (CKY838) and lst4 doa4 (CKY847), bro1 (CKY935) and lst4 bro1 (CKY936), did4 (CKY839) and lst4 did4 (CKY844), vps4 (CKY836) and lst4 vps4 (CKY845), vps27 (CKY837) and lst4 vps27 (CKY846), were spotted as serial dilutions onto minimal ammonia medium, with or without a sub-LC of ADCB (7 mg/l). As control strains, a wild-type strain (CKY835) and the mutants lst4 (CKY695), bul1 bul2 (CKY698), and lst4 bul1 bul2 (CKY699) grown in identical conditions, are shown in the top panel. (B) The same strains growing in minimal urea medium were assayed for [14C]citrulline uptake. Averaged data are expressed as in Figure 1.

lst4

lst4 vps4, lst4 vps27,

lst4 did4,

View larger version (49K): [in this window] [in a new window]   Figure 5. Class E VPS mutants accumulate Gap1p-GFP in endosomal and perivacuolar membranes in the presence of glutamate or an lst4 mutation. (A) Fluorescence microscopy images of wild-type cells (CKY834) and the mutants lst4 (CKY848), vps4 (CKY850), vps27 (CKY851), did4 (CKY849), bro1 (CKY937), lst4 vps4 (CKY854), lst4 vps27 (CKY855; leucine auxotrophy covered by the CEN-LEU2 plasmid pRS415), lst4 did4 (CKY853), lst4 bro1 (CKY938), expressing genomic GFP-tagged Gap1p and exponentially growing in minimal ammonia medium, or incubated for 1 h in the presence of 3 mM glutamate before imaging. (B) A subset of class E vps mutations causes accumulation of Gap1p-GFP in endosomal and perivacuolar membranes in the absence of high concentrations of amino acids. Cells from wild-type strain (CKY835) and the mutants vps27 (CKY837), did2 (CKY857), hse1 (CKY858), vps23 (CKY859), vps37 (CKY860), and vps60 (CKY861), expressing Gap1p-GFP from a centromeric plasmid (pGAP1-GFP) and exponentially growing in minimal ammonia medium were imaged by fluorescence microscopy.

bul1 bul2

lst4

Different Subsets of MVE Proteins Have Different Effects on the Retention of Gap1p in the MVE
Our results above indicate that a specific subset of class E vps mutants growing in the absence of high amino acid concentrations fail to retain Gap1p in the MVE. Although all class E vps mutations showed increased levels of Gap1p activity under these conditions, some of the mutants had more subtle effects on Gap1p. Mutants such as vps60 and hse1 were slightly less sensitive to low concentrations of ADCB (our unpublished data). Similarly, did2, hse1, vps23, and vps37, had only 5- to 7-fold increased levels of Gap1p activity when grown in urea as a nitrogen source (Table 4), which is about half of the effect shown by the mutations described above.

We examined the subcellular localization of Gap1p-GFP in this different subgroup using did2, hse1, vps23, vps37, or vps60 null mutant strains. These single mutant strains growing in ammonia displayed a pattern of Gap1p-GFP localization similar to that of a vps27 lst4 double mutant (compare Figure 5, A and B), with most of the Gap1p-GFP being located intracellularly. Confocal microscopy of these cells revealed that the diffuse pattern of localization of Gap1p-GFP was the result of some Gap1p-GFP in punctate structures adjacent to the vacuole as well as in the vacuolar membrane (our unpublished data). Apparently mutations in this subset of ESCRT genes do not allow Gap1p to exit the MVE efficiently. The differences among ESCRT mutants with respect to their effect on Gap1p do not strictly correlate with the known subdivisions of these proteins into different complexes; however, most of the mutants that allow efficient cycling of Gap1p to the plasma membrane are in ESCRT complex II and III.

A Preexisting Pool of Gap1p Can Cycle from the MVE to the Plasma Membrane
To demonstrate directly the existence of a pathway for cycling of existing Gap1p from the MVE to the plasma membrane, we accumulated Gap1p in the class E compartment in a vps4 mutant grown on glutamate and then transferred the cells to amino acid–free medium. As expected, the vps4 mutant grown on glutamate exhibited low levels of Gap1p activity, but when these cells were transferred to urea medium Gap1p activity increased rapidly (Figure 6A). The initial rise in activity experienced by the vps4 mutant did not depend on newly synthesized permease because the early phase of induction of activity was not blocked by addition of 1.5 µg/ml cycloheximide. This concentration of cycloheximide was chosen as the minimum necessary to inhibit translation completely as assayed by incorporation of radiolabeled methionine into newly translated proteins (Figure 6B; Roberg et al., 1997a). In contrast, activity did not increase substantially in an lst4 vps4 double mutant (that was not treated with cycloheximide), showing that Gap1p cycling to the plasma membrane does depend on Lst4p activity (Figure 6A). An immunoblot control (Figure 6C) shows that each of the cultures contained similar amounts of Gap1p protein, indicating that the differences in activity were not due to Gap1p degradation. In a parallel localization experiment, Gap1p-GFP could be seen to partially redistribute from the endosome to the plasma membrane in a vps4 mutant after transfer from glutamate to urea medium. However, most of the Gap1p-GFP remained in the class E compartment in an lst4 vps4 double mutant under the same conditions (Figure 6D).

View larger version (28K): [in this window] [in a new window]   Figure 6. Gap1p accumulated in the prevacuolar compartment of a class E vps mutant can recycle to the plasma membrane. (A) The strains vps4 (CKY836) and vps4 lst4 (CKY845), grown in minimal glutamate medium, were transferred to minimal urea medium and immediately assayed for [14C]citrulline uptake at different times after the shift. Activity time courses are represented as filled diamonds (vps4) or filled triangles (vps4 lst4). The strain vps4 was also assayed in the presence of 1.5 µg/ml cycloheximide in the urea-containing medium before shift, to inhibit translation of newly synthesized Gap1p (). (B) The effect of cycloheximide (0, 1.5, 10, and 100 µg/ml) on bulk translation was assayed by pulse labeling vps4 (CKY836) on minimal glutamate medium with [35S]methionine. (C) Protein extracts taken at the same time-point periods and using the same strains and conditions as in A, were subject to SDS-PAGE and Western blotting with Gap1p antibody. As a loading control, Pgk1p levels are shown (bottom blot). Each lane contains an extract from the same number of cells. (D) The strains vps4 (CKY850) and lst4 vps4 (CKY854) were monitored for Gap1p-GFP localization at different periods of time after being shifted from minimal glutamate medium to urea medium. For comparison, images of the same strains steadily growing in urea are also shown.

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Gap1p Recycling Does Not Depend on Other Known Pathways for Recycling Proteins from the Endosome to Golgi Compartments
Three different protein complexes have been identified for their role in the recycling of different subsets of proteins from the endosome to the Golgi. The vps fifty three (VFT)/Golgi-associated retrograde protein (GARP) complex consists of four subunits (Vps51p–Vps54p) and is required in association with the Rab Ypt6p for the retrograde transport of Golgi resident proteins from the endosome to the Golgi (Conibear and Stevens, 2000; Siniossoglou and Pelham, 2002). A second complex, known as the retromer complex, is necessary for recycling of Vps10p and is composed of Vps35p, Vps29p, and Vps26p and the Vps5p–Vps17p sorting nexin dimer (Reddy and Seaman, 2001; and reviewed by Seaman, 2005). A third multisubunit tethering complex with a recently discovered role in the recycling of the uracil permease Fur4p (Bugnicourt et al., 2004) that was first identified as being involved in the docking and fusion of vacuolar and/or endosomal membranes is the class C Vps/homotypic fusion and vacuole protein sorting (HOPS) complex (reviewed by Wickner, 2002; Whyte and Munro, 2002). Proteins forming this complex include Pep3/Vps18p, Pep5/Vps11p, Vps16p, Vps33p, Vam3p, and Ypt7 (Raymond et al., 1992, Whyte and Munro, 2002). Notably, a requirement for Vam3p in the recycling of Gap1p has been suggested (Nikko et al., 2003).

If any of these complexes were necessary for Gap1p trafficking from the MVE to the plasma membrane, the corresponding mutants would be expected to exhibit an effect on Gap1p trafficking similar to lst4. However, none of the mutations in the three known recycling complexes exhibited a significant decrease in Gap1p activity (Tables 3 and 4; Figure 7). Moreover, some of the class C Vps mutant strains, even showed increased levels of Gap1p activity that closely resembled to those observed for class E Vps mutations.

View larger version (17K): [in this window] [in a new window]   Figure 7. Recycling of Gap1p is not severely affected by defects in pathways involved in the recycling of proteins from the MVE. The following strains were grown in liquid minimal urea medium and assayed for [14C]citrulline uptake: wild type (CKY835), lst4 (CKY695), vps4 (CKY836), lst4 vps4 (CKY845), vps5 (Y01845), vps5 vps4 (CKY995), vps26 (Y01370), vps26 vps4 (CKY996), vps51 (Y05091), vps51 vps4 (CKY997), vps52 (Y04318), vps52 vps4 (CKY998), vps16 (Y02783), vps16 vps4 (CKY999), vps18 (Y04105), vps18 vps4 (CKY1000), ypt6 (Y05171), ypt6 vps4 (CKY1001), ypt7 (Y00575), ypt7 vps4 (CKY1002), vam3 (Y02362), vam3 vps4 (CKY1003), vps33 (Y05305), vps33 vps4 (CK1004). Measurements of Gap1p activity are expressed as in Figure 1. Single mutant strains in the BY4741 background (EUROSCARF deletion strains), and some of the double mutant strains were transformed with the centromeric plasmid pCEN-HIS3-LEU2-MET15 (pCK283) to eliminate auxotrophic requirements for amino acids.

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Specific Dependence of Gap1p Recycling on LST4 and LST7 Genes
We were interested in whether the genes we have identified that do have a significant effect on Gap1p trafficking from the MVE to the plasma membrane affected other recycling processes. We therefore examined LST4 and LST7, two of the genes we have isolated that have the greatest effect on Gap1p trafficking to the plasma membrane (Roberg et al., 1997b), for their effects on a variety of trafficking events. Both lst4 and lst7 caused a greater than 20-fold decrease in Gap1p activity in the context of a vps4 genetic background (Figure 8A). This decreased Gap1p activity did not correspond to a decrease in GAP1 transcription (Figure 8B) and most likely resulted from a failure of Gap1p to be transported efficiently from the class E compartment to the plasma membrane (Figure 8C). However, neither lst4 nor lst7 appeared to have an effect on Vps10p cycling between the Golgi and endosome because these mutants did not cause an increase in secretion of pro-CPY into the extracellular medium (Figure 8D). Moreover, neither lst4 nor lst7 had an effect on FM4-64 recycling from an endosomal compartment back to the plasma membrane (Figure 8, E and F). Taken together these results indicate that Gap1p has a discrete set of genetic requirements for trafficking from the MVE to the plasma membrane that does not appear to overlap with known endosomal or MVE recycling pathways.

View larger version (32K): [in this window] [in a new window]   Figure 8. LST4 and LST7 are required specifically for Gap1p sorting. (A) lst4 and lst7 mutations interfere with Gap1p distribution to the plasma membrane caused by a vps4 mutation. The following strains were grown in minimal urea medium and assayed for uptake of [14C]citrulline and [14C]arginine: wild type (CKY835), lst4 (CKY695), lst7 (CKY994), vps4 (CKY836), lst4 vps4 (CKY845), and lst7 vps4 (CKY1005). (B) The decrease in Gap1p activity caused by lst4 and lst7 mutations is not a transcriptional effect. Wild type (CKY835), lst4 (CKY695), and lst7 (CKY994) strains were transformed with the PGAP1-LacZ (pMS29) plasmid and assayed for -galactosidase activity after growth in minimal urea medium. (C) lst4 and lst7 mutations cause constitutive sorting of Gap1p to the vacuole and block its recycling caused by a vps4 mutation. Cells from wild-type strain (CKY835) and the mutants lst4 (CKY695), lst7 (CKY994), lst4 vps4 (CKY845), and lst7 vps4 (CKY1005), expressing Gap1p-GFP from a centromeric plasmid (pGAP1-GFP) and exponentially growing in minimal ammonia medium were visualized by fluorescence microscopy. (D) Wild-type (CKY835) vps4 (CKY836), lst4 (CKY695), and lst7 (CKY994) were spotted on a nitrocellulose membrane on YPD plates (an equivalent to 0.5 OD600 of cells/spot) and after 16 h at 30°C secreted CPY was detected using monoclonal anti-CPY (Molecular Probes). (E and F) lst4 and lst7 mutations do not interfere with recycling of FM4-64. (E) The wild-type strain BY4741 was assayed for the ability to recycle the fluorescent membrane dye FM4–64 as explained in Materials and Methods. Fluorescence was measured every second for 10 min on a spectrofluorometer and the graphic represents the average of three independent experiments. As a negative control the same strain was treated with 10 mM NaN3 to block vesicular trafficking, and as a positive control the mutant strain from identical genetic background, rcy1 (Y01221) was also assayed. (F) The same experiment as in E was carried out in parallel in the wild-type (CKY835), lst4 (CKY695), and lst7 (CKY994) strains.

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View larger version (36K): [in this window] [in a new window]   Figure 9. A lst4 mutation or growth on glutamate causes redistribution of Gap1p independently of endocytosis. Fluorescence microscopy images from cells of the wild-type (CKY834) strain, the mutant lst4 (CKY848), and the mutants impaired in endocytosis end3 (CKY874), and end3 lst4 (CKY875), expressing genomic GFP-tagged Gap1p, are shown. Cells were continuously grown to exponential phase in minimal medium with ammonia (top panel) or glutamate (bottom panel) as the only nitrogen source. Images of cells taken 30 min after the addition of glutamate 3 mM to induce endocytosis of Gap1p-GFP are also shown (middle panel). (B) The same strains were grown in liquid minimal ammonia medium and assayed for [14C]citrulline and [14C]arginine uptake, and the data were averaged as in Figure 1.

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View larger version (35K): [in this window] [in a new window]   Figure 10. Constitutive vacuolar sorting of Gap1p in a lst4 mutant does not depend on GGA function. The following strains were transformed with the centromeric plasmid (pGAP1-GFP) and imaged by fluorescence microscopy to detect