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Vol. 18, Issue 1, 295-312, January 2007

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Endocytic Recycling in Yeast Is Regulated by Putative Phospholipid Translocases and the Ypt31p/32p–Rcy1p Pathway

Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, Sapporo, 060-0815, Japan

Submitted May 26, 2006; Revised October 25, 2006; Accepted October 26, 2006
Monitoring Editor: Sean Munro

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phospholipid translocases (PLTs) have been implicated in the generation of phospholipid asymmetry in membrane bilayers. In budding yeast, putative PLTs are encoded by the DRS2 gene family of type 4 P-type ATPases. The homologous proteins Cdc50p, Lem3p, and Crf1p are potential noncatalytic subunits of Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively; these putative heteromeric PLTs share an essential function for cell growth. We constructed temperature-sensitive mutants of CDC50 in the lem3 crf1 background (cdc50-ts mutants). Screening for multicopy suppressors of cdc50-ts identified YPT31/32, two genes that encode Rab family small GTPases that are involved in both the exocytic and endocytic recycling pathways. The cdc50-ts mutants did not exhibit major defects in the exocytic pathways, but they did exhibit those in endocytic recycling; large membranous structures containing the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor Snc1p intracellularly accumulated in these mutants. Genetic results suggested that the YPT31/32 effector RCY1 and CDC50 function in the same signaling pathway, and simultaneous overexpression of CDC50, DRS2, and GFP-SNC1 restored growth as well as the plasma membrane localization of GFP-Snc1p in the rcy1 mutant. In addition, Rcy1p coimmunoprecipitated with Cdc50p-Drs2p. We propose that the Ypt31p/32p–Rcy1p pathway regulates putative phospholipid translocases to promote formation of vesicles destined for the trans-Golgi network from early endosomes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Most eukaryotic cells display an asymmetric distribution of phospholipids in the plasma membrane. In general, the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) are enriched in the inner leaflet facing the cytoplasm, whereas phosphatidylcholine (PC), sphingomyelin, and glycolipids are predominantly found in the outer leaflet of the plasma membrane. This lipid asymmetry is generated and maintained by ATP-driven lipid transporters or translocases. The type 4 subfamily of P-type ATPases is implicated in the translocation of phospholipids from the external to the cytosolic leaflet (Graham, 2004; Pomorski et al., 2004; Holthuis and Levine, 2005). Five members of this subfamily (Drs2p, Neo1p, Dnf1p, Dnf2p, and Dnf3p) are encoded by the genome of the yeast Saccharomyces cerevisiae (Catty et al., 1997). Dnf1p and Dnf2p are localized to the plasma membrane, and loss of Dnf1p and Dnf2p abolishes ATP-dependent transport of fluorescently 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)–labeled analogues of PE, PS, and PC from the outer to the inner plasma membrane leaflet (Pomorski et al., 2003). Drs2p is localized to endosomes and the trans-Golgi network (TGN) (Chen et al., 1999; Hua et al., 2002; Pomorski et al., 2003; Saito et al., 2004), suggesting that Drs2p regulates phospholipid asymmetry in these membranes. Moreover, Golgi membranes isolated from a temperature-sensitive drs2 mutant lacking DNF1, DNF2, and DNF3 exhibited defects in the ATP-dependent transport of an NBD-labeled analogue of PS (Natarajan et al., 2004). Alder-Baerens et al. (2006) also demonstrated that post-Golgi secretory vesicles contained Drs2p- and Dnf3p-dependent NBD-labeled phospholipid translocase activity and that the asymmetric PE arrangement in these vesicles was disrupted in the drs2 dnf3 mutant. The drs2 mutant exhibits TGN defects comparable with those exhibited by strains with clathrin mutations and is defective in the formation of clathrin-coated vesicles (Chen et al., 1999; Gall et al., 2002). Drs2p is also implicated in endocytic recycling because the exocytic vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Snc1p accumulated intracellularly in the drs2 mutant (Hua et al., 2002; Saito et al., 2004).

Cdc50p, a conserved integral membrane protein, was identified as a protein required for polarized growth (Misu et al., 2003). Cdc50p colocalizes with Drs2p at endosomal and TGN membranes, and it associates with Drs2p as a potential noncatalytic subunit (Saito et al., 2004). In the absence of Cdc50p, Drs2p is retained in the endoplasmic reticulum (ER) and vice versa. In addition to Cdc50p, two Cdc50p-related proteins, Lem3p/Ros3p and Crf1p, are encoded by the yeast genome. Similar to Cdc50p, Lem3p is a potential noncatalytic subunit that associates with Dnf1p (Saito et al., 2004) and Dnf2p (see Results), and Crf1p is a potential noncatalytic subunit for Dnf3p (see Results). These heteromeric putative phospholipid translocases (PLTs) constitute an essential family for cell growth in which Cdc50p-Drs2p, Lem3p-Dnf1p/2p, and Crf1p-Dnf3p play major, intermediate, and minor roles, respectively (Hua et al., 2002; Saito et al., 2004). The dnf1 dnf2 drs2 mutant exhibits a defect in endocytic internalization at 15°C as assayed by uptake of the endocytic tracer dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) (see Materials and Methods) (Pomorski et al., 2003). The drs2 dnf1 mutant exhibits a substantial defect in the transport of alkaline phosphatase to the vacuole (Hua et al., 2002). The dnf1 dnf2 dnf3 mutant intracellularly accumulates Snc1p due to defects in endocytic recycling (Hua et al., 2002). The cellular functions shared by these heteromeric putative PLTs, however, need to be further explored.

In this study, we constructed temperature-sensitive (ts) cdc50 mutations in a strain lacking LEM3 and CRF1. Screening for multicopy suppressors of the ts growth phenotype identified YPT32 encoding a Rab family small GTPase, which has been implicated in the formation of exocytic vesicles from the TGN along with its close homologue Ypt31p (Benli et al., 1996; Jedd et al., 1997). The cdc50-ts lem3 crf1 mutants, however, did not exhibit major defects in the formation of exocytic vesicles, but instead they exhibited severe defects in endocytic recycling. Interestingly, during the course of this study, it was reported that Ypt31p/32p also regulate endocytic recycling through its effector Rcy1p (Chen et al., 2005). The F-box protein Rcy1p (recycling 1) is involved in recycling out of early endosomes (Wiederkehr et al., 2000). Rcy1p associates with Skp1p, a component of the Skp1p-cullin-F-box protein (SCF) complex but not with other components required for ubiquitin ligase activity, suggesting that Rcy1p regulates endocytic recycling independently of ubiquitination (Galan et al., 2001). The rcy1 mutant accumulates large membranous structures that seem to be swollen early endosomes (Wiederkehr et al., 2000) and similar structures accumulated in the cdc50-ts lem3 crf1 mutants. Simultaneous overexpression of Cdc50p-Drs2p and GFP-Snc1p suppressed the defects in endocytic recycling of the rcy1 mutant, and Rcy1p was coimmunoprecipitated with Cdc50p and Drs2p. We propose that heteromeric putative PLTs cooperate with Ypt31p/32p-Rcy1p in endocytic recycling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Media and Genetic Techniques
Unless otherwise specified, strains were grown in rich medium (YPDA: 1% yeast extract [Difco, Detroit, MI], 2% bacto-peptone [Difco], 2% glucose, and 0.01% adenine). Strains carrying plasmids were selected in synthetic medium (SD) containing the required nutritional supplements (Rose et al., 1990). When indicated, 0.5% casamino acids (Difco) were added to the SD media without uracil (SDA-Ura). Phosphate-depleted rich medium contained 1% yeast extract, 2% bacto-peptone, and 4% glucose, and it was depleted of phosphate as described by Rubin (1973). The synthetic minimal (MV) media used for cell labeling were prepared as described previously (Rothblatt and Schekman, 1989). Standard genetic manipulations of yeast were performed as described previously (Guthrie and Fink, 1991). Yeast transformations were performed by the lithium acetate method (Elble, 1992; Gietz and Woods, 2002). Escherichia coli strains DH5 and XL1-Blue were used for construction and amplification of plasmids.

Strains and Plasmids
Yeast strains used in this study are listed in Table 1. The cdc50-ts strains were constructed as follows. First, random mutations in CDC50 were introduced by a polymerase chain reaction (PCR)-based method as described previously (Toi et al., 2003) by using the template pKT1262 (YCplac111-CDC50) and the primers CDC50-5' (5'-AAGTGACGAATGGAATGATC-3') corresponding to the nucleotide positions –613 to –594 of CDC50 and CDC50-3'100R (5'-GTCGCACTATTTTCCAAGCG-3') complementary to the nucleotide positions 110–129 downstream of the CDC50 stop codon, to generate the 1.9-kilobase DNA fragment CDC50*. The marker fragment His3MX6 cassette flanked by sequences around the 130 base pairs downstream of the CDC50 stop codon were generated by PCR by using the template pFA6a-His3MX6 (Longtine et al., 1998) and the primers CDC50-3'100F1 (5'-CGCTTGGAAAATAGTGCGACCGGATCCCCGGGTTAATTAA-3') and CDC50-3'100R1 (5'-TTCTATCATTTCATCATCTAAATGGGAATAGCAAACCCTGGGAGTTCTTTGAATTCGAGCTCGTTTAAAC-3') to generate CDC50-3'-His3MX6. The underlined sequence within CDC50-3'100F1 corresponds to the nucleotide positions 110-129 downstream of the CDC50 stop codon, and is complementary to the sequence of CDC50-3'100R; the underlined sequence within CDC50-3'100R1 is complementary to the nucleotide positions 130–179 downstream of the CDC50 stop codon. Then, a second PCR was performed to connect the marker fragment to the randomly mutagenized CDC50 fragment, by using CDC50* and CDC50-3'-His3MX6 as templates and CDC50-5' and CDC50-3'100R1 as primers. The amplified DNA fragment was introduced into the genome of YKT496 (MAT lem3::TRP1), and His⁺ transformants were selected at 25°C. Of these transformants, 418 clones were streaked on two YPDA plates, one plate of which was incubated at 25°C and the other plate was incubated at 37°C. Fourteen clones, which exhibited mild ts growth phenotypes, were crossed with YKM48 (MATa GAL1p-HA-CDC50::KanMX6 crf1::hphMX3), and the resulting diploids were sporulated and dissected. The obtained spore clones whose genotypes were cdc50*::His3MX6 lem3::TRP1 crf1::hphMX3 were tested for growth at 25 and 37°C. The two clones (cdc50-11::His3MX6 lem3::TRP1 crf1::hphMX3 and cdc50-162::His3MX6 lem3::TRP1 crf1::hphMX3) that exhibited the tightest ts phenotype were chosen for further analyses.

drs2 dnf3 DNF2-EGFP

drs2 dnf3 DNF2

drs2 dnf3 dnf2

drs2

rcy1::KanMX4

lem3::KanMX4

rcy1::hphMX4

rcy1::KanMX4

vps1

vps4

vps30

The plasmids used in this study are listed in Table 2. The YPT32^Q72L and YPT32^S27N alleles were made by using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the plasmid pKT1555 (YEplac181-YPT32). The mutagenic oligonucleotides were YPT32-Q72L-1 (GAT TTG GGA CAC GGC AGG TCT AGA ACG TTA CAG GGC CAT CAC G) and its complement YPT32-Q72L-2 for YPT32^Q72L and YPT32-S27N-1 (GAC TCC GGT GTG GGT AAA AAT AAT CTG TTG TCG AGA TTT ACA AC) and its complement YPT32-S27N-2 for YPT32^S27N. We sequenced the entire open reading frame of YPT32 in each construct to verify that only the desired substitution was introduced. Epitope-tagged Rcy1 proteins were functional, because both pRS416-GFP-RCY1 and YEplac195-HA-BS-RCY1 plasmids suppressed the growth defect of the rcy1 mutant at 18°C (our unpublished data). Schemes detailing the construction of plasmids are available upon request.

Isolation of Multicopy Suppressors of the cdc50-ts lem3 crf1 Mutants

cdc50-11 lem3 crf1

cdc50-162 lem3 crf1

Purification of Secretory Vesicles and Enzyme Assays
Purification of secretory vesicles was performed as described (Harsay and Bretscher, 1995) with minor modifications. Briefly, cells were grown at 25°C to the early to mid-logarithmic phase (OD₆₀₀ of 0.5–0.7) in 1 liter of synthetic medium, harvested by centrifugation, and resuspended in phosphate-depleted rich medium. Growth was continued at 25°C for 90 min to induce acid phosphatase secretion. Cells were then incubated at 37°C for 2 h before harvesting. Cells were converted to spheroplasts and lysed using 20 strokes in a Dounce glass homogenizer with a tight pestle (Wheaton Industries, Millville, NJ) in lysis buffer containing 0.8 M sorbitol, with 10 mM triethanolamine and 1 mM EDTA, adjusted to pH 7.2 with acetic acid (TEA), and protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). A 700 x g spin for 10 min generated the pellet (P1) and supernatant (S1) fractions. The S1 fraction was spun at 13,000 x g for 20 min to generate P2 and S2. The S2 fraction was centrifuged at 100,000 x g for 1 h in an SW28 rotor (Beckman Coulter, Fullerton, CA) to generate membrane pellets (P3). P3 membrane pellets were overlaid with 300 µl of 0.8 M sorbitol/TEA and placed on ice for 2–3 h to allow easy resuspension. For gradient fractionation, a 12-ml 15–30% continuous Nycodenz gradient in 0.8 M sorbitol/TEA was created. The P3 membranes were adjusted to 35% Nycodenz in a total volume of 1 ml and loaded into the bottom of the gradient using a 10-cm needle. Gradients were centrifuged in a P40ST rotor (Hitachi, Tokyo, Japan) at 100,000 x g for 19 h, and 0.8-ml fractions were manually collected from the top of the samples.

Enzymatic assays for acid phosphatase, exoglucanase, and invertase activities were performed as described previously (Harsay and Bretscher, 1995), except that fractions were not diluted when the invertase activity was measured.

Antibodies
The rabbit anti-Pma1p polyclonal antibodies were gifts from R. Serrano (Polytechnic University of Valencia, Valencia, Spain). The rabbit anti-carboxypeptidase Y (CPY) and anti-invertase polyclonal antibodies were gifts from A. Nakano (RIKEN, Wako, Japan). Rabbit anti-Lem3p polyclonal antibodies were gifts from M. Umeda (Kyoto University, Kyoto, Japan). The mouse anti-HA (HA.11) and anti-myc (9E10) monoclonal antibodies were purchased from BAbCO (Richmond, CA) and Sigma-Aldrich (St. Louis, MO), respectively. The horseradish peroxidase-conjugated secondary antibodies (sheep anti-mouse IgG and donkey anti-rabbit IgG) used for immunoblotting were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom).

Immunoprecipitation Analysis
Immunoprecipitation analysis of Lem3p-Dnf2p and Crf1p-Dnf3p was performed as described previously (Saito et al., 2004). Coimmunoprecipitation analysis of Rcy1p with Cdc50p, Drs2p, Dnf1p, and Dnf2p was similarly performed. Briefly, cells were grown at 30°C to a cell density of 0.5 OD₆₀₀/ml in SDA-Ura medium. Cells collected from 400 ml of the cultures were washed twice with ice-cold water and resuspended at 200 OD₆₀₀/ml in lysis buffer (10 mM Tris-HCl, pH 7.5, 0.3 M sorbitol, 0.1 M NaCl, and 5 mM MgCl₂) containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). The cells were lysed by Multi-beads shocker (Yasui Kikai, Osaka, Japan). Cell lysates were centrifuged at 400 x g for 5 min, and the resulting supernatant was centrifuged at 100,000 x g for 1 h at 4°C. For immunoprecipitation, the pellets were solubilized in 0.8 ml of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, and 1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate [CHAPS]) containing the above-mentioned protease inhibitors. Insoluble material was removed by centrifugation at 20,630 x g for 5 min at 4°C. The cleared lysates were incubated with 5 µg of anti-Myc antibody for 1 h at 4°C. We then rotated the samples with 20 µl of protein G-Sepharose 4 Fast Flow (GE Healthcare) for 1 h at 4°C. The protein G-Sepharose beads were pelleted and washed three times with immunoprecipitation buffer in the absence of detergents before suspending them in SDS-PAGE sample buffer.

Immunoblotting Analysis
Immunoblotting analysis was performed as described previously (Misu et al., 2003). For SDS-PAGE of Drs2p-Myc, Dnf1p-Myc, Dnf2p-Myc, and Dnf3p-Myc, samples were heated at 37°C for 20 min before loading. For SDS-PAGE of Pma1p, 10-µl aliquots of each fraction from the Nycodenz density gradient fractionation were mixed with 10 µl of 2X SDS-PAGE sample buffer, followed by heating at 37°C for 15 min before loading. Anti-HA, anti-Myc, and anti-Lem3p antibodies were used at a 1:1000 dilution. Anti-Pma1p antibodies were used at a 1:5000 dilution. For the colony blot assay, anti-CPY antibodies were used at a 1:1000 dilution.

Microscopic Observations
Cells were observed using a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF superhigh-pressure mercury lamp and a 1.4 numerical aperture 100x Plan Apo oil immersion objective lens (Nikon Instec) with appropriate fluorescence filter sets (Nikon Instec) or differential interference contrast optics. Images were acquired using a cooled digital charge-coupled device camera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) and AQUACOSMOS software (Hamamatsu Photonics). Observations are based on the characterization of >100 cells.

To assess the endocytic pathway using the lipophilic styryl dye FM4-64, cells were grown to late-logarithmic phase in YPDA medium at 25°C or 37°C for 3 h. Four OD₆₀₀ units of cells were labeled with 32 µM FM4-64 (Invitrogen, Carlsbad, CA) in 100 µl of YPDA medium for 15 min at 25 or 37°C. Cells were harvested by centrifugation, resuspended in 200 µl of fresh YPDA medium, and chased at 25 or 37°C for 30 min. Cells were washed twice with 100 µl of SD medium and immediately observed using a G-2A filter set. To mark early endosomes with FM4-64, cells were grown to early logarithmic phase in SDA-Ura medium at 25°C and then shifted to 37°C for 3 h. Four OD₆₀₀ units of cells were incubated with 32 µM FM4-64 in 100 µl of YPDA medium on ice for 30 min. Cells were harvested by centrifugation, resuspended in 200 µl of fresh YPDA medium and chased at 37°C for 10 min. After chase, cells were harvested by centrifugation, resuspended in ice-cold SD medium, and immediately observed. To visualize the vacuole lumen, cells were stained with CellTracker Blue CMAC (Invitrogen) according to the manufacturer's protocol and observed using a UV-1A filter set.

Most GFP- or mRFP1-tagged proteins were observed in living cells, which were grown to early to mid-logarithmic phase, harvested, and resuspended in SD medium. Cells were immediately observed using a GFP bandpass (for GFP) or a G2-A (for mRFP1) filter set. Localization of GFP- or mRFP1-tagged proteins was also examined in fixed cells. Fixation was performed for 5 min at 37°C by direct addition of a commercial 37% formaldehyde stock (Wako Pure Chemical Industries, Osaka, Japan) to a final concentration of 0.5% in the medium, followed by a 10-min incubation at 25°C. After fixation, cells were washed twice with phosphate-buffered saline and examined. The cell fixation protocol resulted in images of the GFP- and mRFP1-fusion proteins that were similar to those obtained with living cells (our unpublished observation).

Electron Microscopy (EM)
Ultrastructural observation of cells by conventional EM was performed using the glutaraldehyde-permanganate fixation technique (Kaiser and Schekman, 1990). Cells were embedded in Spurr's resin (Nissin EM, Tokyo, Japan). Thin sections (50–60 nm) were cut on an Ultracut microtome (Leica, Wetzlar, Germany) equipped with a Sumiknife (Sumitomo Electric Industries, Osaka, Japan), stained with 3% uranyl acetate and Reynold's lead citrate, and viewed using an H-7100 electron microscope (Hitachi) at 75 kV. Immuno-EM was performed using the aldehyde fixation/metaperiodate permeabilization method (Mulholland and Botstein, 2002), except that glutaraldehyde was not included in the fixative. Cells were embedded in LR White resin (medium grade; London Resin Company, Berkshire, United Kingdom) and sectioned as described above. Mouse anti-HA antibody (HA.11) was used as a primary antibody at a 1:500 dilution. Ten-nanometer gold-conjugated anti-mouse IgG antibodies (BBInternational, Cardiff, United Kingdom) were preadsorbed with fixed wild-type cells and used as secondary antibodies at a 1:100 dilution. Samples were poststained with uranyl acetate and viewed as described above.

³⁵S Pulse-Chase and Immunoprecipitation Experiments
Metabolic labeling of yeast cells, preparation of cell extracts, and immunoprecipitation were performed essentially as described previously (Rothblatt and Schekman, 1989; Gaynor et al., 1994; Yahara et al., 2001). To visualize target proteins, total proteins or immunoprecipitated proteins were resolved by SDS-PAGE followed by analysis using a FLA3000 fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan).

To assay internal and external invertase, cells were grown to mid-logarithmic phase in MV-low sulfate medium containing 5% glucose at 37°C for 2 h. Three (labeled for 7 min) or five (labeled for 2 min) OD₆₀₀ units of cells were collected, converted to spheroplasts with Zymolyase 100T (25 U/ml cell suspension; Seikagaku Kogyo, Tokyo, Japan), and incubated at 37°C for 30 min. Spheroplasts were resuspended in MV-no sulfate medium containing 1 M sorbitol and 0.1% glucose, grown at 37°C for 30 min, and labeled with 50 µCi of Tran³⁵S-label (PerkinElmer Life and Analytical Sciences, Boston, MA) per OD₆₀₀ unit of cells for 7 or 2 min. A chase was initiated by adding a chase solution (MV-no sulfate medium with 4 mM ammonium sulfate, 0.012% L-cysteine, and 0.016% L-methionine) containing 0.1% glucose and 1 M sorbitol to an equal volume of the samples. After the pulse-chase, ice-cold sodium azide was added to spheroplast suspensions to a final concentration of 10 mM, and the suspensions were separated by centrifugation into spheroplasts and media as the intracellular and extracellular fractions, respectively. Both fractions were subjected to immunoprecipitation using rabbit anti-invertase antibodies.

To assay total proteins secreted into the medium, cells were grown to mid-logarithmic phase in MV-low sulfate medium containing 5% glucose at 37°C for 2 h. Three OD₆₀₀ units of cells were collected, resuspended in MV-no sulfate medium containing 2% glucose and 0.03% bovine serum albumin, and grown at 37°C for 30 min. The cells were labeled with 100 µCi of Tran³⁵S-label per OD₆₀₀ unit of cells at 37°C for 15 min. A chase was initiated by adding the chase solution containing 2% glucose and 0.5% casamino acids to an equal volume of the samples. After a 45-min chase, cell suspensions were treated with sodium azide and separated into intracellular and extracellular fractions as described above. Trichloroacetic acid (TCA) was added to both fractions to a final concentration of 10%, and the fractions were kept on ice for 15 min, followed by centrifugation at 20,630 x g for 15 min. After one wash with 10% TCA and two washes with ice-cold acetone, pellets were solubilized in SDS-PAGE sample buffer.

To examine the sorting pathway for CPY, cells were pulse chased as described above. After a chase, cells were suspended in spheroplast stop solution (1 M sorbitol, 25 mM Tris-HCl, pH 7.5, 20 mM NaN₃, 10 mM dithiothreitol, and 20 mM NaF) and treated with 2.5 U/ml Zymolyase 100T. Intracellular and extracellular fractions were prepared as in the assay for invertase and subjected to immunoprecipitation by using rabbit anti-CPY antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cdc50 Family Proteins Form Complexes with Drs2 Family Proteins
In a previous study, we demonstrated that Cdc50p and Lem3p associate with Drs2p and Dnf1p, respectively (Saito et al., 2004). Dnf1p and its close homologue Dnf2p (83% similar and 69% identical at the amino acid sequence level) exhibit redundant activities in phospholipid translocation across the plasma membrane (Pomorski et al., 2003; our unpublished data). Therefore, we examined whether Lem3p physically associates with Dnf2p in coimmunoprecipitation experiments. We constructed a gene that encoded a C-terminally Myc-tagged version of Dnf2p and introduced it into cells. Dnf2p-Myc was immunoprecipitated with an anti-Myc antibody from membrane protein extracts prepared by solubilization in 1% CHAPS. Lem3p was coimmunoprecipitated with Dnf2p-Myc, whereas Lem3p was not detected in immunoprecipitates from cells lacking DNF2-Myc (Figure 1A). In addition, Dnf2p-EGFP as well as Dnf1p-EGFP was localized in the ER in the lem3 mutant (Figure 1B). Neither Cdc50p nor Crf1p-HA was coimmunoprecipitated with Dnf2p-Myc, and Dnf2p-EGFP was localized to the plasma membrane in cdc50 and crf1 mutants (our unpublished data). Both Crf1p and Dnf3p colocalize with markers for early/late endosomes or the TGN, and deletion of either CRF1 or DNF3 resulted in no discernible phenotypes (Hua et al., 2002; Pomorski et al., 2003; our unpublished observation). We examined whether Crf1p binds to Dnf3p in coimmunoprecipitation experiments. We created cells expressing both a C-terminally Myc-tagged version of DNF3 and an HA-tagged version of CRF1. Crf1p-HA was coimmunoprecipitated with Dnf3p-Myc, whereas Crf1p-HA was not detected in immunoprecipitates from cells lacking DNF3-Myc (Figure 1A). In addition, Dnf3p-EGFP was localized in the ER of the crf1 mutant (Figure 1C). Neither Cdc50p nor Lem3p was coimmunoprecipitated with Dnf3p-Myc, and the localization patterns of Dnf3p-EGFP in cdc50 and lem3 mutants were identical to that in wild-type cells (our unpublished data). In addition, neither Drs2p-Myc nor Dnf1p-Myc coimmunoprecipitated with Crf1p-HA, and the localization patterns of Drs2p-EGFP and Dnf1p-EGFP in the crf1 mutant were identical to that in wild-type cells (our unpublished data). These results, together with our previous study (Saito et al., 2004), demonstrate that Cdc50 family proteins (Cdc50p, Lem3p, and Crf1p) form complexes with Drs2 family proteins (Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively) in vivo.

View larger version (61K): [in this window] [in a new window]   Figure 1. Cdc50 family proteins form complexes with Drs2 family proteins. (A) Coimmunoprecipitation of Lem3p and Crf1p with Dnf2p and Dnf3p, respectively. Cells were grown at 25°C to a cell density of 0.5 OD600/ml in YPDA medium. Membrane extracts were then prepared as described in Materials and Methods. Myc-tagged Dnf2p or Dnf3p was immunoprecipitated with an anti-Myc antibody from these extracts. Immunoprecipitates were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against Lem3p or HA (top) and Myc (bottom). The results shown are representative of several experiments. The strains used were as follows: YKT1099 (DNF2-Myc LEM3) and YKT1098 (DNF2 LEM3) (left) and YKT1100 (DNF3-Myc CRF1-HA) and YKT1098 (DNF3 CRF1-HA) (right). (B) Localization of Dnf2p-EGFP. Wild-type (YKT921) and lem3 (YKT923) cells containing the DNF2-EGFP construct in the genome were grown to early to mid-logarithmic phase in YPDA medium at 30°C and immediately observed by fluorescence microscopy. (C) Localization of Dnf3p-EGFP. Wild-type (YKT925) and crf1 (YKT928) cells containing the DNF3-EGFP construct in the genome were grown and observed as described in (B). Bars, 5 µm.

lem3 crf1

cdc50-11 lem3 crf1

cdc50-162 lem3 crf1

View larger version (45K): [in this window] [in a new window]   Figure 2. Overexpression of YPT32 or YPT31 suppresses the temperature-sensitive growth defect of the cdc50-ts mutants. (A) The domain structure of Cdc50p and the amino acid substitutions in Cdc50-11p and Cdc50-162p. The black boxes indicate potential transmembrane domains. (B) Suppression of the cdc50-ts mutations by multicopy YPT32 or YPT31. cdc50-11 (YKT993) and cdc50-162 (YKT942) cells were transformed with pKT1555 (YEplac181-YPT32), pKT1554 (YEplac181-YPT31), pKT1259 (YEplac181-CDC50), or a control vector (YEplac181). Transformants were streaked onto a YPDA plate, followed by incubation at 37°C for 2 d. (C) The GTP-bound form, but not the GDP-bound form, of Ypt32p suppresses the ts growth defect of cdc50-ts mutants. cdc50-11 (YKT993) and cdc50-162 (YKT942) mutant cells were transformed with pKT1578 (YEplac181-YPT32Q72L), pKT1579 (YEplac181-YPT32S27N), pKT1555 (YEplac181-YPT32), or a control vector (YEplac181). For each plasmid, four independent transformants were streaked onto a YPDA plate, followed by incubation at 37°C for 3 d.

cdc50

To obtain clues to essential functions of the CDC50 and DRS2 families, we screened for multicopy suppressors of the growth defect of the cdc50-ts mutants at 37°C. We found that overexpression of YPT32 suppressed, albeit weakly, the growth defect of the cdc50-ts mutants (Figure 2B). Ypt32p, a Rab family small GTPase, shares a high degree of sequence similarity (81% identity and 90% similarity) with Ypt31p, overproduction of which also weakly suppressed the growth defect of the cdc50-ts mutants (Figure 2B). Overexpression of YPT32^Q72L, which mimics a GTP-bound form of the protein, but not of YPT32^S27N, which mimics a GDP-bound form, suppressed the ts growth phenotype of the cdc50-ts mutants to the same extent as overexpression of YPT32 (Figure 2C), suggesting that YPT32 suppressed the cdc50-ts mutations by hyperactivation of its downstream pathway through its effectors. Previous studies have reported that YPT31/32 are involved in intra-Golgi transport, the formation of transport vesicles at the TGN (Benli et al., 1996; Jedd et al., 1997), and the recruitment of Sec2p, the guanine nucleotide exchange factor for Sec4p, to secretory vesicles (Ortiz et al., 2002). Therefore, we investigated the vesicular trafficking phenotypes in the cdc50-ts mutants.

The Secretory Pathway Seems Normal in a cdc50-ts Mutant
Because YPT31/32 have been shown to be required for exit from the TGN in the exocytic pathway (Benli et al., 1996; Jedd et al., 1997) and the drs2 mutant exhibits defects in Golgi function (Chen et al., 1999; Hua et al., 2002), we investigated defects in the secretory pathways of the cdc50-11 mutant. We first tested for the secretion of the periplasmic enzyme invertase. Because invertase acquires N-linked oligosaccharide chains, which are heterogeneously modified in the Golgi, it is secreted as a highly glycosylated protein and thus occurs as a high-molecular-mass smear after SDS-PAGE. In secretory mutants, invertase secretion is inhibited and the enzyme accumulates in the lumen of secretory compartments. Cells were incubated at 37°C for 2.5 h, induced to produce invertase for 30 min, pulse labeled for 7 min with Tran³⁵S-label, and chased at 37°C. After 15 min of chase, wild-type cells secreted most of the invertase as a fully glycosylated form. In the cdc50-11 mutant, invertase was also secreted in a fully glycosylated form with kinetics similar to those of wild-type cells, whereas with a control secretory mutant, sec2-56 (Novick et al., 1980), invertase remained intracellular even after a 30-min chase (Figure 3A). To examine a kinetic delay of invertase secretion more precisely, cells were pulse labeled for 2 min and chased for 2, 5, 10, and 15 min. In the cdc50-11 mutant, invertase was secreted in a fully glycosylated form with kinetics similar to that of wild-type cells after a 10-min chase, although a slight delay was observed in the external appearance of invertase after a 5-min chase (Figure 3B).

View larger version (72K): [in this window] [in a new window]   Figure 3. Secretion and formation of secretory vesicles are nearly normal in a cdc50-ts mutant. (A and B) Pulse-chase experiments of invertase secretion. Wild-type (YKT38), cdc50-11 (YKT993), and sec2-56 (ANS2-3A) cells (A) or wild-type (YKT38) and cdc50-11 (YKT993) cells (B) were grown for 2.5 h at 37°C, induced to produce invertase for 30 min, pulse labeled with Tran35S-label for 7 min (A) or 2 min (B), and chased for the indicated time at 37°C. Samples were separated by centrifugation into internal (I) and external (E) fractions. Invertase was recovered by immunoprecipitation and visualized by SDS-PAGE and a phosphorimager system. (C) General secretion by the cdc50-11 mutant. Wild-type (YKT38), cdc50-11 (YKT993), and sec2-56 (ANS2-3A) cells were grown for 2.5 h at 37°C, pulse labeled with Tran35S-label for 15 min, and chased for 45 min. Samples were separated by centrifugation into I and E fractions. Proteins in each fraction were precipitated with TCA and visualized by SDS-PAGE and a phosphorimager system. (D) Western blots for Pma1p, a marker for low-density vesicles, in Nycodenz gradient fractions. sec6-4 (YKT1010) and cdc50-11 sec6-4 (YKT1011) cells transformed with pKT1486 (PACT1-SUC2) were incubated for 2 h at 37°C. Secretory vesicles were prepared and fractionated as described in Materials and Methods. Numbered fractions (as indicated at the top) were heated in sample buffer for 15 min at 37°C, separated by SDS-PAGE, and probed with antibodies against Pma1p. (E) Activities of marker enzymes for high-density vesicles in Nycodenz gradient fractions. Fractions were prepared from sec6-4 (diamonds) and cdc50-11 sec6-4 (triangles) cells as described in D. Hydrolyzing enzyme activities are expressed in arbitrary units based on the absorbance measured at 415 nm (acid phosphatase, top; exoglucanase, middle) or at 540 nm (invertase, bottom).

Harsay and Bretscher (1995) used the ts late-acting sec mutant sec6-4 to demonstrate that newly synthesized secretory proteins are exported via at least two different classes of secretory vesicles, which have similar diameters, but differ in their densities. The high-density vesicles (HDSVs) contain the soluble, secreted enzymes invertase and acid phosphatases together with most of the exoglucanase activity, whereas the low-density vesicles (LDSVs) contain the plasma membrane H⁺-ATPase Pma1p (Harsay and Bretscher, 1995; David et al., 1998). Disruption of a gene encoding a dynamin-related protein (VPS1) or the clathrin heavy chain (CHC1) abolishes the production of HDSVs, yielding LDSVs that contain all the secreted cargo (Gurunathan et al., 2002). Because DRS2 was implicated in the formation of clathrin-coated vesicles (Chen et al., 1999; Gall et al., 2002), we examined whether the production of these two types of secretory vesicles was affected in the cdc50-11 mutant. We created a quadruple cdc50-11 lem3 crf1 sec6-4 mutant (cdc50-11 sec6-4 mutant) and purified secretory vesicles by Nycodenz density gradient centrifugation of membranes prepared from temperature-shifted cdc50-11 sec6-4, or sec6-4 mutant cells. Western blots of the gradient fractions indicated that Pma1p levels were highest in the lower density membranes from the cdc50-11 sec6-4 mutant as well as from the sec6-4 mutant (Figure 3D), whereas the activities of invertase, acid phosphatase, and exoglucanase were found in the higher density fractions from both the cdc50-11 sec6-4 and sec6-4 mutants (Figure 3E). We also carried out the density gradient purification of membranes from sec6-4 and cdc50-11 sec6-4 cells grown at the permissive temperature (25°C). Pma1p was not detected by Western blots of any gradient fractions from sec6-4 and cdc50-11 sec6-4 cells (our unpublished data). Similarly, activities of invertase, acid phosphatase, and exoglucanase were not found in any fractions from both the sec6-4 and sec6-4 cdc50-11 mutants (our unpublished data). These results suggested that the activities of invertase, acid phosphatase, and exoglucanase and the Pma1p levels observed in the density gradient fractions from both strains incubated at 37°C reflected the high- and low-density classes of exocytic vesicles, respectively. These results suggest that the cdc50-11 mutant did not exhibit major defects in production of these two types of secretory vesicles.

The Endocytic Pathway to the Vacuole and the Vacuolar Protein Sorting Pathway Seems Normal in the cdc50-ts Mutants
Endocytic transport from the plasma membrane to the vacuole was previously examined in dnf and drs2 mutants. Defective endocytic internalization of the lipophilic dye FM4-64 and -mating factor was demonstrated in the dnf1 dnf2 drs2 mutant (Pomorski et al., 2003), whereas the drs2, cdc50, and dnf1 dnf2 dnf3 mutants were shown to be defective in a certain step in the postinternalization transport to the vacuole (Chen et al., 1999; Hua et al., 2002; Misu et al., 2003). All these assays were conducted at lower temperatures such as 15 or 18°C. The cdc50-ts mutants, however, internalized and delivered FM4-64 to the vacuole at 37°C during a 30-min incubation (Figure 4A). Although the sizes of the vacuoles visualized by FM4-64 staining were somewhat smaller in the cdc50-ts mutants, this phenotype was not affected by the incubation temperature. These results suggest that the essential function of the putative PLTs at higher temperatures is not in endocytic transport from the plasma membrane to the vacuole.

View larger version (83K): [in this window] [in a new window]   Figure 4. Normal endocytic and VPS pathways in the cdc50-ts mutants. (A) Internalization and transport of FM4-64 to the vacuole. Wild-type (YKT38), cdc50-11 (YKT993), and cdc50-162 (YKT942) cells grown for 3 h were stained with FM4-64 for 15 min in YPDA medium, and chased for 30 min in fresh YPDA medium, at the indicated temperature. Bar, 5 µm. (B) Pulse-chase experiments of CPY intracellular transport. Wild-type (YKT38) and cdc50-11 (YKT993) cells were grown for 2.5 h at 37°C, pulse labeled with Tran35S-label for 15 min, and chased for 45 min at 37°C. CPY was immunoprecipitated from I and E fractions, resolved by SDS-PAGE, and visualized using a phosphorimager system. (C) Secretion of CPY. Wild-type (YKT38), cdc50-11 (YKT993), vps1 (KKT276), vps4 (KKT277), and vps30 (AKY15) cells were grown for 3 h at 37°C in contact with a nitrocellulose filter, and secreted CPY was detected by probing with antibodies against CPY. (D) Localization of Vps10p-EGFP. Wild-type (YKT957), cdc50- 11 (YKT1086), and cdc50-162 (YKT1088) cells containing the VPS10-EGFP construct in the genome were grown for 3 h at 25 or 37°C in SD medium. Bar, 5 µm. (E) Localization of GFP-Pep12p. Wild-type (YKT38), cdc50-11 (YKT993), and cdc50-162 (YKT942) cells transformed with pKT1487 (pRS416-GFP-PEP12) were grown for 3 h at 25°C or 37°C in SDA-Ura medium. Bar, 5 µm.

drs2, cdc50

dnf1 drs2

vps1, vps30

vps4

The membrane-bound CPY-sorting receptor Vps10p has been reported to cycle between the TGN and late endosomes (Cereghino et al., 1995; Cooper and Stevens, 1996), and a complex called the "retromer," which comprises five proteins, is required for the endosome-to-TGN retrieval of Vps10p (Seaman et al., 1997). Thus, the removal of components of the retromer results in missorting of Vps10p to the vacuole (Seaman et al., 1997). We created cells expressing a C-terminally EGFP-tagged version of VPS10 and observed the localization of Vps10p-EGFP. In wild-type cells, Vps10p-EGFP resulted in a punctate fluorescence pattern characteristic of TGN-localized yeast proteins (Figure 4D) as described previously (Cereghino et al., 1995; Cooper and Stevens, 1996). Similarly, Vps10p-EGFP created a punctate fluorescence pattern in the cdc50-11 and cdc50-162 mutants incubated at 37°C as well as 25°C (Figure 4D), suggesting that Vps10p-EGFP was normally retrieved from late endosomes in the cdc50-ts mutants. These results are consistent with our observation that CPY sorting was normal in the cdc50-11 mutant (Figure 4, B and C).

We also analyzed the localization of the endosomal syntaxin Pep12p. Whereas endogenous Pep12p typically occurs as scattered or perivacuolar puncta (Lewis et al., 2000), GFP-Pep12p, the expression of which was driven by the strong TPI1 promoter, reached the vacuolar outer membrane via late endosomes (Black and Pelham, 2000). We observed the localization of GFP-Pep12p expressed under the control of the TPI1 promoter in the cdc50-ts mutants. In wild-type cells and the cdc50-ts mutants, GFP-Pep12p was found in small punctate structures and vacuolar membranes at 25 and 37°C (Figure 4E). The small punctate structures seemed to be late endosomes, because they were much smaller than the vacuoles. These results suggest that in the cdc50-ts mutants, GFP-Pep12p was normally transported to late endosomes, and overexpressed GFP-Pep12p was further delivered to the vacuole.

Together, our results suggest that the cdc50-ts mutants do not have major defects in the endocytic pathway to the vacuole, the VPS pathway, or the recycling pathway between the TGN and late endosomes. Thus, PLTs composed of proteins from the Cdc50p and Drs2p families are unlikely to be essential for membrane trafficking via these pathways at higher temperatures.

cdc50-ts Mutants Show Mislocalization of Recycling Proteins
During the course of this study, Segev and colleagues reported that Ypt31p/32p are also involved in the retrieval of the furin homologue Kex2p and the v-SNARE Snc1p from endosomes to the TGN (Chen et al., 2005). Thus, we examined localization of GFP-tagged versions of these proteins in the cdc50-ts mutants. Kex2p is a TGN resident protein that is localized due to constant retrieval from late and early endosomes (Brickner and Fuller, 1997; Lewis et al., 2000). At 25°C, C-terminally EGFP-tagged Kex2p resulted in cytoplasmic fluorescent puncta typical of proteins that are localized in the yeast TGN (Figure 5A). We also observed weak fluorescent signals that seem to correspond to vacuoles as judged by colocalization with CellTracker Blue CMAC, a dye that stains the vacuolar lumen (Supplemental Figure S2). In lem3 crf1 mutant cells incubated at 37°C, punctate localization patterns of Kex2p-GFP were observed as in wild-type cells, although they were less clear. In contrast, in the cdc50-ts mutants incubated at 37°C, Kex2p-EGFP was primarily localized in vacuoles (Figure 5A and Supplemental Figure S2), as was demonstrated in the ypt31 ypt32-ts mutant (Chen et al., 2005). Similar to Vps10p, Kex2p mislocalizes to the vacuole when cycling between endosomes and the TGN is impaired (Wilcox et al., 1992; Cooper and Stevens, 1996; Conibear and Stevens, 2000). Because Vps10p exhibited the normal punctate localization in the cdc50-ts mutants (Figure 4D), our results suggest that the cdc50-ts mutants are defective in the retrieval pathway from early endosomes.

View larger version (70K): [in this window] [in a new window]   Figure 5. Recycling marker proteins are mislocalized in the cdc50-ts mutants. (A) Localization of Kex2p-EGFP. Wild-type (YKT903), cdc50-11 (YKT1000), cdc50-162 (YKT1001), and lem3 crf1 (YKT1310) cells containing the KEX2-EGFP construct in the genome were grown for 3 h at 25 or 37°C in SD medium. (B) Localization of GFP-Snc1p. Wild-type (YNF63), cdc50-11 (YNF65), cdc50-162 (YNF67), lem3 crf1 (YNF61), and dnf1 dnf2 dnf3 (YNF784) cells carrying the pRS416-GFP-SNC1 plasmid were grown for 3 h at 25 or 37°C in SDA-Ura medium. Bars, 5 µm.

cdc50, drs2

dnf1 dnf2 dnf3

lem3 crf1

lem3 crf1

lem3 crf1

dnf1 dnf2 dnf3

dnf1 dnf2 dnf3

lem3 crf1

lem3 crf1

dnf1 dnf2 dnf3

The atypical labeling of intracellular structures with GFP-Snc1p in the cdc50-ts mutants could result from defects in the transport pathway from the TGN to the plasma membrane or in that from the plasma membrane to the TGN via endosomes. When cdc50-11 cells preincubated at 37°C for 3 h were treated with 100 µM of the actin assembly inhibitor latrunculin A (LAT-A) for 10 min at 37°C to block endocytosis, GFP-Snc1p was observed to be localized to the plasma membrane in addition to intracellular large structures (Figure 6A); the percentages of cdc50-11 cells with GFP-Snc1p at the plasma membrane increased from 42% (n = 183) (dimethyl sulfoxide [DMSO] control) to 83% (n = 133) (LAT-A treatment). These results suggested that newly synthesized GFP-Snc1p reached the plasma membrane in the mutant cells. Consistent with this result, a mutant form of GFP-Snc1p, GFP-Snc1p-pm, which can reach the plasma membrane but cannot be internalized by endocytosis (Lewis et al., 2000), was exclusively localized to the plasma membrane in the cdc50-11 mutant as well as wild-type cells at 37°C (Figure 6B). These results also suggest that GFP-Snc1p needs to be endocytosed to be incorporated into the large intracellular structures. To confirm this with GFP-fused wild-type Snc1p, cells grown at 25°C were pretreated with 100 µM LAT-A for 10 min at 25°C (0 min), followed by incubation at 37°C for 30 min in the presence of 100 µM LAT-A (30 min) (Figure 6C). LAT-A treatment inhibited the intracellular accumulation of GFP-Snc1p in the cdc50-11 mutant. These results suggest that cdc50-11 mutant cells are defective in the plasma membrane-to-TGN transport pathway but not in the TGN-to-plasma membrane transport pathway.

View larger version (55K): [in this window] [in a new window]   Figure 6. The cdc50-ts mutant is defective in plasma membrane-to-TGN transport. (A) Plasma membrane localization of GFP-Snc1p is restored in the cdc50-ts mutant by blocking endocytosis with LAT-A. Wild-type (YNF63) and cdc50-11 (YNF65) cells carrying the pRS416-GFP-SNC1 plasmid were preincubated at 37°C for 3 h and then treated with 100 µM LAT-A or DMSO (vehicle control) for 10 min at 37°C in SDA-Ura medium. (B) Localization of GFP-Snc1p-pm. Wild-type (YKT38) and cdc50-11 (YKT993) cells transformed with pRS416-GFP-SNC1 pm were grown to early logarithmic phase at 25°C and shifted to 37°C for 3 h in SDA-Ura medium. (C) Pretreatment with LAT-A inhibits abnormal accumulation of GFP-Snc1p in the cdc50-ts mutant. Wild-type (YNF63) and cdc50-11 (YNF65) cells carrying the pRS416-GFP-SNC1 plasmid were grown to early logarithmic phase at 25°C and were then pretreated with 100 µM LAT-A or DMSO (vehicle control) for 10 min at 25°C in SDA-Ura medium (0 min), followed by incubation at 37°C for 30 min in SDA-Ura medium in the presence (LAT-A) or absence (DMSO) of 100 µM LAT-A (30 min). Bars, 5 µm.

Given that Snc1p accumulates in intracellular compartments in ypt31 ypt32-ts mutant cells (Chen et al., 2005), we tested for the effects of overproduction of the GTP-bound (Q72L) or GDP-bound (S27N) form of Ypt32p (Figure 2C) on the localization of GFP-Snc1p. In cells overexpressing YPT32^Q72L, GFP-Snc1p was primarily localized to the plasma membrane of the growing site as it was in wild-type cells. In contrast, GFP-Snc1p accumulated in abnormal compartments located in the bud or near the bud neck in 15% of the cells carrying YPT32^S27N (n = 149) (Supplemental Figure S3), similar to the results observed with the cdc50-ts mutants. Because Ypt31p/32p were implicated in transport out of the TGN (Benli et al., 1996; Jedd et al., 1997), we examined whether GFP-Snc1p-pm reached the plasma membrane in the mutant strains. GFP-Snc1p-pm was exclusively localized to the plasma membrane in cells carrying YPT32^S27N or YPT32^Q72L (Supplemental Figure S3). These results also suggest that YPT32 is involved in endocytic recycling.

We next examined whether overexpression of YPT32 suppressed mislocalization of GFP-Snc1p in the cdc50-ts mutants. When YPT32 was overexpressed at 37°C, the percentages of cdc50-11 and cdc50-162 mutant cells with GFP-Snc1p at sites with polarized plasma membranes increased from 39% (n = 117) to 60% (n = 120) and from 43% (n = 170) to 55% (n = 141), respectively (our unpublished data). Similarly, the percentages of cdc50-11 and cdc50-162 mutant cells with GFP-Snc1p in large compartments at 37°C slightly decreased from 44% (n = 119) to 35% (n = 132) and from 38% (n = 120) to 28% (n = 140), respectively (our unpublished data). These results are consistent with a weak suppression of the growth defect in the cdc50-ts mutants by overexpression of YPT32 (Figure 2B). In addition, overexpression of YPT32 did not suppress the cold-sensitive growth phenotype of the cdc50 mutant at 18°C (our unpublished data). These results suggest that YPT32 is not a bypass suppressor of the cdc50 mutations and thus raise the possibility that YPT32 requires the residual PLT activity of Cdc50p-Drs2p for the suppression.

In the cdc50-11 Mutant, GFP-Snc1p and GFP-Tlg1p Seem to Accumulate in Early Endosome-derived Structures
We morphologically examined the Snc1p-containing structures in the cdc50-11 mutant. To examine whether the large structures were associated with the TGN, the TGN was visualized with a TGN marker, C-terminally mRFP1-tagged Sec7p (Franzusoff et al., 1991), in cdc50-11 mutant cells expressing GFP-SNC1. Although Sec7p-mRFP1 was observed in punctate structures scattered throughout wild-type and cdc50-11 mutant cells at 37°C, few of the GFP-Snc1p–positive structures were colocalized with Sec7p-mRFP1 (Figure 7A), suggesting that the large GFP-Snc1p-labeled structures were not associated with the TGN. We next examined whether a recycling defect in the cdc50-ts mutants caused GFP-Snc1p to be missorted to the vacuole as with Kex2p-EGFP. Few of the GFP-Snc1p–positive structures, however, were also stained by CellTracker Blue CMAC (Figure 7B), suggesting that GFP-Snc1p did not accumulate in the vacuole. Thus, our results suggest that GFP-Snc1p is associated with endosomal structures.

View larger version (60K): [in this window] [in a new window]   Figure 7. GFP-Snc1p and GFP-Tlg1p accumulate in early endosome-derived structures in the cdc50-ts mutant. (A) GFP-Snc1p and the TGN marker Sec7p-mRFP1 are not colocalized in the cdc50-11 mutant. cdc50-11 SEC7-mRFP1 (YNF153) cells carrying pRS416-GFP-SNC1 were incubated at 37°C for 3 h in SDA-Ura medium. Obtained images were merged to compare the two signal patterns. (B) GFP-Snc1p is not localized to vacuoles stained with CellTracker Blue CMAC in the cdc50-11 mutant. cdc50-11 (YNF65) cells carrying the pRS416-GFP-SNC1 plasmid were grown at 37°C for 3 h, followed by staining with 100 µM CellTracker Blue CMAC at 37°C for 15 min. (C) GFP-Snc1p was colocalized with FM4-64 after a short incubation in the cdc50-11 mutant. Wild-type (YNF63) and cdc50-11 (YNF65) cells carrying the pRS416-GFP-SNC1 plasmid were grown at 37°C for 3 h, stained with 32 µM FM4-64 on ice for 30 min, and chased in fresh medium at 37°C for 10 min. (D) mRFP1-Snc1p and GFP-Tlg1p are colocalized in the cdc50-11 mutant. Wild-type (YKT38) and cdc50-11 (YKT993) cells cotransformed with pKT1566 (YEplac181-GFP-TLG1) and pKT1563 (pRS416-mRFP1-SNC1) were incubated at 37°C for 3 h in SD-Leu-Ura medium, followed by microscopic examination after fixation with 0.5% formaldehyde. Bars, 5 µm.

ypt31 ypt32-ts

rcy1

ypt31 ypt32-ts

Because in the rcy1 mutant, the target membrane-associated (t)-SNARE Tlg1p seems to intracellularly accumulate in abnormal compartments (Wiederkehr et al., 2000) as was observed for Snc1p, we examined the localization of Tlg1p in cdc50-11 mutant cells. Tlg1p is recycled between the TGN and early endosomal membrane, and is consequently present in both membranes (Lewis et al., 2000). To examine the localization of Tlg1p in the cdc50-11 mutant, we constructed a multicopy plasmid containing GFP-TLG1 driven by its own promoter. In wild-type cells, GFP-Tlg1p was observed to be scattered throughout the cell (Figure 7D). In contrast, there was a substantial decrease of GFP-Tlg1p localized to punctate structures in cdc50-11 mutant cells, and GFP-Tlg1p was instead localized to large compartments (Figure 7D). To examine whether Tlg1p and Snc1p were localized in the same large compartments in cdc50-11 mutant cells, we constructed a single-copy plasmid containing the mRFP1-SNC1 gene driven by the TPI1 promoter and examined cells coexpressing GFP-TLG1 and mRFP1-SNC1. Quantitative analysis of individual spots revealed that in the cdc50-11 mutant cells, 96% of the mRFP1-Snc1p–positive large structures were also labeled with GFP-Tlg1p (n = 170), whereas 86% of the GFP-Tlg1–positive structures were also labeled with mRFP1-Snc1p (n = 163) (Figure 7D). These results suggest that the cdc50-11 mutant was defective in the retrieval pathway from early endosomes to the TGN and that Snc1p and Tlg1p accumulated in the abnormal structures derived from early endosomes in the cdc50-ts mutants.

Electron Microscopic Examination of the Abnormal Structures Containing HA-Snc1p in the cdc50-11 Mutant
EM was performed to visualize and characterize the abno