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Vol. 15, Issue 7, 3418-3432, July 2004

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Cdc50p, a Protein Required for Polarized Growth, Associates with the Drs2p P-Type ATPase Implicated in Phospholipid Translocation in Saccharomyces cerevisiae

Koji Saito ^*, Konomi Fujimura-Kamada ^*, Nobumichi Furuta ^*, Utako Kato , Masato Umeda , and Kazuma Tanaka ^* 

^* Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-0815, Japan; Division of Molecular Biology and Information, Institute for Chemical Science, Kyoto University, Kyoto 611-0011, Japan

Submitted November 20, 2003; Revised April 5, 2004; Accepted April 8, 2004
Monitoring Editor: Randy Schekman

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cdc50p, a transmembrane protein localized to the late endosome, is required for polarized cell growth in yeast. Genetic studies suggest that CDC50 performs a function similar to DRS2, which encodes a P-type ATPase of the aminophospholipid translocase (APT) subfamily. At low temperatures, drs2 mutant cells exhibited depolarization of cortical actin patches and mislocalization of polarity regulators, such as Bni1p and Gic1p, in a manner similar to the cdc50 mutant. Both Cdc50p and Drs2p were localized to the trans-Golgi network and late endosome. Cdc50p was coimmunoprecipitated with Drs2p from membrane protein extracts. In cdc50 mutant cells, Drs2p resided on the endoplasmic reticulum (ER), whereas Cdc50p was found on the ER membrane in drs2 cells, suggesting that the association on the ER membrane is required for transport of the Cdc50p-Drs2p complex to the trans-Golgi network. Lem3/Ros3p, a homolog of Cdc50p, was coimmunoprecipitated with another APT, Dnf1p; Lem3p was required for exit of Dnf1p out of the ER. Both Cdc50p-Drs2p and Lem3p-Dnf1p were confined to the plasma membrane upon blockade of endocytosis, suggesting that these proteins cycle between the exocytic and endocytic pathways, likely performing redundant functions. Thus, phospholipid asymmetry plays an important role in the establishment of cell polarity; the Cdc50p/Lem3p family likely constitute potential subunits specific to unique P-type ATPases of the APT subfamily.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell polarity is the ultimate manifestation of the complex mechanisms that establish and maintain functionally specialized domains within the plasma membrane and cytoplasm. The asymmetric organization of the cytoskeleton, secretory pathway, and plasma membrane along an appropriate axis is established by specific proteins assembling a polarized and specialized cortical actin cytoskeleton (for review, see Drubin and Nelson, 1996). Polarized actin networks then mediate sorting and delivery of factors required to execute and maintain cell polarity.

The budding yeast Saccharomyces cerevisiae is an excellent model system to study the regulation of cell and cytoskeletal polarity (for reviews, see Pringle et al., 1995; Drubin and Nelson, 1996). During S. cerevisiae budding, the rigid cell wall is expanded locally as a result of polarized secretion. Cell surface extensions are preceded by the polarized organization of actin filament-containing structures, including actin cortical patches and actin cables. Cortical actin patches, small foci of actin filaments and associated proteins, cluster near regions of growth (for review, see Pruyne and Bretscher, 2000). These structures are required during endocytosis for internalization (for reviews, see Geli and Riezman, 1998; Wendland et al., 1998). Actin cables, bundles of actin filaments arising from discrete regions of the plasma membrane coincident with growth sites, extend throughout the cell. These dynamic cables guide the polarized movements of a type V myosin, Myo2p, to deliver secretory vesicles (Govindan et al., 1995; Schott et al., 1999).

Myo3p and Myo5p, S. cerevisiae type I myosins, are components of cortical patches aiding in the assembly of cortical actin patches. We isolated CDC50 as a multicopy suppressor of the temperature-sensitive myo3 myo5-360 mutant (Misu et al., 2003). The cdc50 mutant displays cold-sensitive cell cycle arrest with small buds. Arrested cdc50 cells are large and round, exhibiting depolarization of cortical actin patches and defects in the formation of actin cables. In addition, polarity regulators, Bni1p and Gic1p, are mislocalized in the cdc50 mutant cells. Bni1p, a component of the 12S polarisome, is a yeast counterpart of mammalian formin and is capable of polymerizing actin (Evangelista et al., 1997; Evangelista et al., 2002; Sagot et al., 2002). Gic1p is a downstream effector of the Cdc42p small GTPase, physically and functionally interacting with 12S polarisome components (Brown et al., 1997; Chen et al., 1997; Jaquenoud and Peter, 2000).

Cdc50p, a conserved integral membrane-spanning protein, is localized primarily to late endosomal/prevacuolar compartments, raising the question of how CDC50 controls the localization of polarity regulators. Mutations of LEM3/ROS3, encoding a protein homologous to Cdc50p, confer hypersensitivity to a phosphatidylethanolamine-binding peptide antibiotic, Ro09-0198 (Kato et al., 2002). Interestingly, a mutant of LEM3 also displays marked decreases in internalization of fluorescently 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled analogs of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), but not of phosphatidylserine (PS) (Kato et al., 2002; Hanson et al., 2003). Lem3p is primarily localized to the plasma membrane, suggesting its involvement in the translocation of phospholipids across the plasma membrane.

The role of specific changes in phospholipid composition of intracellular or plasma membranes in the regulation of development or the maintenance of cell polarity remains unknown. Most cell types display an asymmetric distribution of phospholipids across the plasma membrane (Devaux, 1991; Cerbon and Calderon, 1991; Diaz and Schroit, 1996). In general, aminophospholipids PS and PE are enriched in the inner leaflet facing the cytoplasm, whereas PC, sphingomyelin, and glycolipids are predominantly found in the outer leaflet of the plasma membrane. In the human erythrocyte membrane, 80% of total PE and most of PS are found in the inner leaflet, whereas 76 and 82% of total PC and sphingomyelin are found in the outer leaflet, respectively (Rothman and Lenard, 1977). Similarly, in the budding yeast plasma membrane, 85 and 90% of total PE and PS are found in the inner leaflet, respectively (Cerbon and Calderon, 1991). Loss of this asymmetric distribution triggers a variety of intercellular events. Cell surface exposure of PS promotes platelet activation (Rosing et al., 1980) and acts as a signal for the recognition and removal of apoptotic cells by macrophages (Fadok et al., 2000). Lipid asymmetry is generated and maintained by ATP-driven lipid transporters or translocases (Devaux, 1991), a prime candidate aminophospholipid translocase (APT) of which is ATPase II (Zachowski et al., 1989). Molecular cloning of the ATPase II-encoding gene from bovine chromaffin granules revealed it as a member of a previously unrecognized subfamily of P-type ATPases (Tang et al., 1996). Members of this subfamily differ from cation-transporting P-type ATPases because they lack the negatively charged amino acids within the transmembrane segments critical for cation transport.

Of the five members of this subfamily in S. cerevisiae, DRS2, NEO1, DNF1, DNF2, and DNF3 (Hua et al., 2002), the functions of Drs2p are the best characterized. DRS2 was identified as a mutation that is synthetically lethal with a mutation in ARF1, which encodes an ADP-ribosylation factor (ARF) (Chen et al., 1999). ARF is a small GTPase involved in initiating the formation of COPI and clathrin-coated vesicles (CCVs). Drs2p is localized to the trans-Golgi network (TGN). The drs2 mutant exhibits TGN defects comparable with those exhibited by strains with clathrin mutations (Chen et al., 1999). The drs2 mutant also exhibits a defect in APT activity at the plasma membrane (Tang et al., 1996), although this result remains in dispute (Siegmund et al., 1998; Marx et al., 1999). The lack of detectable differences in APT activity in drs2 mutant cells may be due to Drs2p localization to the TGN. In contrast, loss of Dnf1p and Dnf2p abolishes the ATP-dependent transport of NBD-labeled PE, PC, and PS across the plasma membrane (Pomorski et al., 2003). Because Dnf1p and Dnf2p are localized to the plasma membrane, these proteins are likely the P-type ATPases responsible for the translocation of phospholipids at the plasma membrane.

Because Lem3p and Cdc50p are not structurally related to ATPases, it remains unclear whether Lem3p possesses an APT activity, or functions in conjunction with a P-type ATPase or another APT. Here, we show that the cdc50 and drs2 mutants show similar phenotypes, including cold-sensitive growth, depolarization of cortical actin patches and mislocalization of polarity regulators. Although the lem3 or dnf1 mutation does not affect cell growth, they show synthetic lethal interaction with cdc50 and drs2 mutations. Coimmunoprecipitation experiments also demonstrate that Cdc50p and Lem3p associate with Drs2p and Dnf1p, respectively. Cdc50p and Lem3p also are required for proper localization of Drs2p and Dnf1p, respectively. We therefore propose that the Cdc50p/Lem3p family comprises a set of subunits specific to phospholipid-translocating P-type ATPases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Media and Genetic Techniques
Unless otherwise specified, strains were grown in YPDA rich medium (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 SD medium without uracil (SDA-Ura). Standard genetic manipulations of yeast were performed as described previously (Guthrie and Fink, 1991). Yeast transformations were performed using the lithium acetate method (Elble, 1992; Agatep et al., 1998). 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 summarized in Table 1. Yeast strains carrying complete gene deletions (LEM3), 3 x hemagglutinin (HA)-tagged genes (CDC50, LEM3), 13 x Myc-tagged genes (DRS2, DNF1, and NEO1), enhanced green fluorescent protein (EGFP)-tagged genes (DRS2, DNF1, NEO1, CDC50, and SEC7), and the monomeric red fluorescent protein 1 (mRFP1)-tagged SEC7 gene were constructed by polymerase chain reaction (PCR)-based procedures as described previously (Longtine et al., 1998). The vrp1 and vp527 disruption mutants were constructed as described by Mochida et al. (2002) and by Misu et al. (2003), respectively. The drs2 and dnf1 disruption mutants were constructed on our strain background as follows. Regions containing the disruption marker and the flanking sequences were PCR amplified using genomic DNA derived from either the drs2::KanMX4 or dnf1::KanMX4 strains (a gift from C. Boone, University of Toronto, Ontario, Canada) as a template. The amplified DNA fragment was then introduced into the appropriate strain. All constructs produced by the PCR-based procedure were verified by colony-PCR amplification to confirm the replacement occurred at the expected locus. Epitope-tagged Drs2 and Neo1 proteins were deemed functional, because they supported cell growth at all temperatures tested. Epitope-tagged Dnf1 protein was also functional as cdc50 strain with the epitope-tagged Dnf1 protein was viable at permissive temperatures for cdc50 strain, whereas the cdc50 dnf1 strain was inviable at all temperatures (see below; Table 3). The drs2::hphMX4 and dnf1::hphMX4 strains were constructed by replacing the KanMX4 cassette of YKT745 (drs2::KanMX4) and YKT748 (dnf1::KanMX4), respectively, with the hphMX4 cassette from pAG32 (Goldstein and McCusker, 1999). The drs2 strain expressing BNI1-EGFP and drs2 strain expressing GIC1-EGFP were constructed by crosses of YKT745 (drs2::KanMX4) with YKT455 (BNI1-EGFP::KanMX6) and YKT579 (GIC1-EGFP::KanMX6), respectively, followed by tetrad dissection. The drs2 strain expressing either BNI1-EGFP or GIC1-EGFP was confirmed by colony-PCR amplification.

The plasmid used to construct the SEC7-mRFP1::TRP1 strain was created by PCR amplification of the mRFP1 gene by using the oligonucleotide primers PAmRFP1 (5'-GATTTAATTA ACATGGCCTC CTCCGAGGAC-3') and mRFP1AS (5'-CTTGGCGCGC CTAGGCGCCG GTGGAGTG-3') with the mRFP1 gene within the pRSET_B plasmid (Campbell et al., 2002) as a template. The amplified fragment, followed by digestion with AscI and PacI, was cloned into the AscI-PacI gap of pFA6a-GFP-TRP1 (Longtine et al., 1998) to obtain a plasmid, designated pFA6a-mRFP1-TRP1, in which the original GFP is replaced with mRFP1. pRS416 GFP-SNC1 was generously provided by M. Lewis and H. Pelham (Medical Research Council) (Lewis et al., 2000). The plasmids used in this study are summarized in Table 2. Schemes detailing the construction of plasmids are available upon request.

Isolation of Multicopy Suppressors of the cdc50 Mutant
The cdc50 strain (YKT442) was transformed with a yeast genomic DNA library constructed in the YEp24 multicopy plasmid. As spontaneous revertants often arise in the cdc50 strain at the restrictive temperature (18°C), a homozygous diploid strain was used for the multicopy suppressor screening. After transformation, cells were first incubated at 30°C for 24 h to allow recovery, and then incubated at 18°C for 6 d on SDA-Ura plates. Approximately 570,000 transformants were screened; 41 transformants reproducibly grew at 18°C. The transformants containing CDC50 on the plasmid were identified by colony PCR and eliminated. Plasmids were obtained from each of the remaining transformants for further analysis. All of them conferred cold temperature-resistant growth to YKT442. Plasmids carrying the LEM3 gene were identified by PCR and eliminated. The genes contained within the remaining seven plasmids were identified by sequencing both ends of the inserts, and then classified into three nonoverlapping groups. The group providing the strongest suppressor activity was analyzed. Deletion analysis revealed this suppressor gene as NEO1.

Antibodies
Mouse anti-HA (HA.11) and anti-Myc (9E10) monoclonal antibodies were purchased from Babco (Richmond, CA) and Sigma-Aldrich (St. Louis, MO), respectively. Rabbit anti-Lem3p polyclonal antibodies have been described previously (Kato et al., 2002). The horseradish peroxidase (HRP)-conjugated secondary antibodies (sheep anti-mouse IgG and donkey anti-rabbit IgG) used for immunoblotting were purchased from Amersham Biosciences (Piscataway, NJ). Cy3-conjugated secondary antibodies (donkey anti-mouse IgG) used for immunofluorescence were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Immunoprecipitation and Western Blotting
The preparation of crude membrane and soluble fractions was performed as described previously (Chang and Slayman, 1991) with the following modifications. Cells were grown at 25°C for 12 h to a cell density of 0.5 OD₆₀₀/ml in YPDA medium. Cells collected from 400 ml of cultures were washed three times in lysis buffer (10 mM Tris-HCl, pH 7.5, 0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl₂) and resuspended at 200 OD₆₀₀/ml in lysis buffer containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 mM benzamidine, 1 mM phenylmetylsulfonylfluoride). The cells were then lysed by agitation six times with glass beads on a Vortex mixer for 30 s. Cell lysates were centrifuged at 400 x g for 5 min to remove unbroken cells; the supernatant was then centrifuged at 100,000 x g for 1 h at 4°C (TLA120.2 rotor; Beckman Coulter, Fullerton, CA). The supernatant was removed to obtain a pellet of total membranes. For immunoprecipitation, membrane pellets were solubilized in 0.8 ml of immunoprecipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% 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 split into two aliquots; each was incubated with 5 µg of either anti-Myc antibody or control mouse IgG for 1 h at 4°C. We then rotated these samples with 20 µl of protein G-Sepharose 4 Fast Flow (Amersham Biosciences AB, Uppsala, Sweden) for 1 h at 4°C. The protein G-Sepharose beads were then pelleted and washed three times with immunoprecipitation buffer in the absence of detergents. The immunoprecipitates were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Membranes were blocked in 5% skim milk in 50 mM Tris-HCl, pH 7.5, 200 mM NaCl with 0.05% Tween 20 (TBST) for 30 min at room temperature. Membranes were then incubated with primary antibody (anti-Myc antibody diluted 1:1000, anti-HA antibody diluted 1:1000, or anti-Lem3p antibodies diluted 1:1000 in 5% skim milk in TBST) at 4°C overnight. After three washes with 5% skim milk in TBST, the membrane was incubated in secondary antibody (anti-mouse IgG-HRP or anti-rabbit IgG-HRP diluted 1:1000 in TBST) for 1 h at room temperature. After three washes in TBST, membranes were visualized by chemiluminescence (Amersham Biosciences). Protein bands were detected using LAS1000plus (Fuji Film, Tokyo, Japan), and the signal intensity of the bands was quantified by Science Lab 2001 Image Gauge, version 4.0 (Fuji Film).

Lipid Preparation
1-Palmitoyl-2-(6-NBD-aminocaproyl)-PE (NBD-PE), 1-palmitoyl-2-(6-NBD-aminocaproyl)-PC (NBD-PC), 1-palmitoyl-2-(6-NBD-aminocaproyl)-PS (NBD-PS), and dioleoylphosphatidylcholine (DOPC) were obtained from Avanti Polar Lipids (Alabaster, AL). To prepare large unilamellar vesicles, lipids were mixed at 40 mol % NBD-PE, -PC, or -PS, and 60 mol % DOPC. Chloroform was removed by evaporation followed by vacuum desiccation. Desiccated phospholipids were solubilized in SD medium; the mixture was passed seven times through a LiposoFast-Basic stabilizer (Avanti, Ottawa, Canada), equipped with 0.1-µm filters to produce evenly sized vesicles containing a 1 mM total concentration of lipids.

Internalization of Fluorescence-labeled Phospholipids into Yeast Cells
Fluorescently labeled phospholipids internalization experiments were performed as described by Kato et al. (2002). Briefly, cells carrying the designated plasmids were grown to early-mid logarithmic phase in SD-Ura media at 25°C. After dilution to 0.35 OD₆₀₀/ml in SD-Ura, cells were incubated with vesicles containing 40% NBD-phospholipids and 60% DOPC at a final concentration of 50 µM, shaking for 30 min at 25°C. Cells were then suspended in SD medium containing 0.01% NaN₃ and 2.5 µg/ml propidium iodide (PI) to allow the exclusion of PI-positive dead cells in flow cytometric analysis. Flow cytometry of NBD-labeled cells was performed on a FACS Calibur cytometer by using CellQuest software (BD Biosciences, San Jose, CA). Green fluorescence of the NBD was plotted on a histogram to allow calculation of the mean fluorescence intensity.

Microscopic Observations
Cells were observed using a Nikon ECRIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with a HB-10103AF super high-pressure mercury lamp and a 1.4 numerical aperture 100x Plan Apo oil immersion objective (Nikon Instec) with the appropriate fluorescence filter sets (Nikon Instec) and differential interference contrast (DIC) optics. Images were acquired with a digital cooled charge-couple device camera (C4742-95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) by using AQUACOSMOS software (Hamamatsu Photonics). Observations are compiled from the examination of at least 100 cells.

To visualize GFP-tagged proteins in living cells, cells were grown to earlymid logarithmic phase, harvested, and resuspended in SD medium. Cells were mounted on microslide glass and observed immediately using a GFP (green) bandpass filter set. To observe Bni1p-GFP and Gic1p-GFP, cells were fixed for 10 min at 18°C by direct addition of 37% formaldehyde (Wako Pure Chemicals, Osaka, Japan) to a final concentration of 2% as described previously (Kawasaki et al., 2003). Images of GFP-fusion proteins in fixed cells were similar to those observed in living cells.

To observe filamentous actin, cells were grown to early logarithmic phase, fixed in formaldehyde, and stained with tetramethylrhodamine isothiocyanate-phalloidin (Sigma-Aldrich) as described previously (Mochida et al., 2002). After six washes in phosphate-buffered saline, cells were mounted in 90% glycerol containing n-propyl gallate (Wako Pure Chemicals). Cells were immediately observed microscopically using a G-2A (red) bandpass filter set.

Lypophilic styryl dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) (Molecular Probes, Eugene, OR) staining was performed as described previously (Misu et al., 2003). Cells were grown to late logarithmic phase in YPDA medium at 30°C. Four OD₆₀₀ units of cells were incubated with 32 µM FM4-64 at 30°C for 15 min in 100 µl of YPDA medium. Cells were harvested by centrifugation, resuspended in 200 µl of fresh YPDA medium, and chased at 30°C for 30 min. After the chase period, cells were washed twice in SD medium and immediately observed microscopically using a G-2A bandpass filter set.

The preparation of cells for Lem3p-HA immunofluorescence analysis was performed as described previously (Berkower et al., 1994). Cells were grown to mid-logarithmic phase in YPDA medium at 30°C. Cells were fixed in 4% formaldehyde at 30°C for 20 min, permeabilized, and incubated with a mouse anti-HA monoclonal antibody (HA.11) diluted at 1:2000. Antibody binding was visualized by treatment with Cy3-conjugated anti-mouse IgG antibodies diluted at 1:500 (Jackson ImmunoResearch Laboratories). After four washes in PBS, cells were mounted in 90% glycerol containing n-propyl gallate (Wako Pure Chemicals). Cells were observed microscopically using a G-2A bandpass filter set.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Isolation of NEO1 as a Multicopy Suppressor of the cdc50 Mutation
To identify genes involved in the regulation or function of Cdc50p, we isolated multicopy suppressors of the cold-sensitive growth phenotype of cdc50 mutant at 18°C, and found NEO1 in those genes. Overexpression of NEO1 also suppressed the defects in actin cytoskeletal organization in the cdc50 mutant at 18°C. It increased both cell populations with polarized cortical actin patches and actin cables from <5% to 30% of small-budded cdc50 mutant cells (our unpublished data). NEO1 was originally isolated as a gene conferring resistance to the aminoglycoside neomycin upon overexpression (Prezant et al., 1996). The NEO1 gene, a member of the yeast DRS2/NEO1 family, encodes a P-type ATPase belonging to the APT subfamily (Catty et al., 1997). Other members of this family, Dnf1p, Dnf2p, and Drs2p, regulate transbilayer phospholipid arrangement (Pomorski et al., 2003). Overexpression of DNF1 also partially suppressed a cold-sensitive growth defect of the cdc50 mutant at 18°C (our unpublished data).

In yeast, Cdc50p has two structural homologues, Lem3p/Ros3p and Ynr048wp. Here, we designate the YNR048w gene CRF1 (CDC50/ROS3 family 1). A triple null mutant of CDC50, LEM3, and CRF1 is not viable. Overexpression of either LEM3 or CRF1 suppresses the cold-sensitive growth defect of the cdc50-1 mutant (Radji et al., 2001), demonstrating that these three genes constitute an essential gene family with substantial functional overlaps. We also isolated LEM3 as a multicopy suppressor of the cold-sensitive growth defect of the cdc50 mutant at 18°C (see MATERIALS AND METHODS). Recent observations that the lem3 mutant displays defects in the internalization of fluorescently labeled phospholipids (Kato et al., 2002; Hanson et al., 2003) suggest that Cdc50p and Crf1p have similar biochemical functions. The CDC50/LEM3 family may be functionally related to DRS2/NEO1 family, although these two families are structurally unrelated. We therefore investigated the functional relationship between the CDC50/LEM3 and DRS2/NEO1 families.

Genetic Interaction between CDC50/LEM3 Family and DRS2/NEO1 Family
We examined the growth profile of cells lacking two of the CDC50/LEM3 family members; the cdc50 lem3 mutant exhibited a severe growth defect, whereas the cdc50 crf1 mutant exhibited a similar cold-sensitive growth defect as seen in the cdc50 mutant. The lem3 crf1 mutant grew as well as wild-type cells (our unpublished data). These results indicate that CDC50 and LEM3 have substantial functional overlap in cell growth regulation, whereas CRF1 contributes little in the absence of CDC50 or LEM3. The investigation of Hua et al. (2002) into the genetic interactions among the members of the DRS2/NEO1 family (NEO1, DRS2, DNF1, DNF2, and DNF3) revealed that NEO1 is an essential gene with unique functions. The drs2 mutant exhibits a cold-sensitive growth phenotype, whereas dnf1, dnf2, or dnf3 single mutants grow normally at all temperatures tested. A dnf1 dnf2 dnf3 triple mutant also grows normally, at similar rate as seen for wild-type cells. The drs2 dnf1 mutant, however, displays a severe growth defect, whereas neither dnf2 nor dnf3 mutations exacerbated the growth phenotype of the drs2 mutant. These results indicate that DRS2 and DNF1 likely overlap in their functions during cell growth, whereas DNF2 and DNF3 contribute little to cell growth in the absence of DRS2.

We therefore investigated the genetic interactions between the CDC50/LEM3 and DRS2/DNF1 genes, which are the members of the CDC50/LEM3 and DRS2/NEO1 families that contribute most substantially to cell growth. We generated strains carrying combinations of these null alleles and analyzed growth at various temperatures (Table 3). The cdc50 and drs2 mutants display similar growth phenotypes; both strains grew normally at 30°C or higher temperatures, grew slowly at 25°C, and did not grow at all at 18°C. Interestingly, both cdc50 dnf1 and drs2 lem3 double mutants exhibited synthetic growth defects. Furthermore, neither the cdc50 drs2 nor the dnf1 lem3 double mutants displayed reduced growth rates in comparison with respective single mutants. These results suggest that DRS2 and CDC50 perform similar functions, whereas DNF1 and LEM3 share similar gene functions, in spite of the lack of structural similarity between the DRS2/NEO1 and CDC50/LEM3 families.

The drs2 Mutant Shows Defects in Polarized Growth
As the cdc50 mutant exhibits defects in the establishment of cell polarity (Misu et al., 2003), we examined whether the drs2 mutant shows similar defects. drs2 mutant cells were grown at 18°C for 12 h as described previously (Misu et al., 2003) and analyzed for morphology and the distribution of filamentous actin. The cdc50 mutant cells exhibited a large and round morphology at 18°C (Figure 1A; Misu et al., 2003). drs2 mutant cells also were large and round at 18°C, although the magnitude of this effect was less pronounced than that seen in the cdc50 mutant (Figure 1A). Another morphological phenotype of cdc50 mutants is that cdc50 cells are arrested with small buds (Moir et al., 1982; Misu et al., 2003). The fraction of small-budded cells increased from 25% at 30°C to 40% after incubation at 18°C for 12 h (Misu et al., 2003). In contrast, no increase of small-budded cells could be observed for drs2 cells; when grown at either 18 or 30°C, drs2 mutant cell cultures contained 29 or 30% cells with small buds, respectively. Wild-type cells, in a manner similar to the drs2 mutant, contained 31% cells with small buds at both 18 and 30°C. cdc50 mutant cells also exhibited depolarization of cortical actin patches and defects in formation of actin cables (Misu et al., 2003). The depolarization of cortical actin patches could be observed in >90% of the small-budded cdc50 mutant cells at 18°C (Figure 1A; Misu et al., 2003). Staining of filamentous actin with phalloidin revealed that cortical actin patches were depolarized in 50% of the small-budded drs2 mutant cells at 18°C, in contrast to 8% of the wild-type cells with small buds (Figure 1A). When grown at 30°C, actin patches were depolarized in 13% of the small-budded drs2 mutant cells and only 4% of the wild-type cells with small buds. Actin cables were absent from >95% of the small-budded drs2 mutant cells at 18°C, as well as cdc50 mutant cells (Figure 1A; Misu et al., 2003). At 30°C, actin cables were normally formed and polarized properly in >95% of the small-budded drs2 mutants and wild-type cells. These results suggest that the drs2 mutant is defective in the establishment of cell polarity, but exhibits less severe phenotypes than the cdc50 mutant.

View larger version (72K): [in this window] [in a new window]   Figure 1. drs2 and cdc50 mutants exhibit defects in polarized growth. (A) Organization of the actin cytoskeleton in drs2 and cdc50 cells. Wild-type (YKT39), cdc50 (YKT249), and drs2 (YKT745) strains were grown in YPDA medium for 12 h at 18 or 30°C. After fixation, cells were stained with tetramethylrhodamine isothiocyanate-phalloidin and visualized by either DIC or epifluorescence. Bar, 5 µm. (B) Localization of GFP-tagged Bni1 and Gic1 proteins in drs2 and cdc50 cells. Cells were grown in YPDA medium for 12 h at 18°C and observed after fixation in 2% formaldehyde. The strains used were as follows: YKT455 (Bni1p-GFP in WT), YKT495 (Bni1p-GFP in cdc50), YKT779 (Bni1p-GFP in drs2), YKT579 (Gic1p-GFP in WT), YKT580 (Gic1p-GFP in cdc50), and YKT781 (Gic1p-GFP in drs2). Bar, 5 µm.

Bni1p and Gic1p, regulators of polarized growth, are normally localized to growing sites, such as a bud tip or a cell division site. In the cdc50 mutant, both of these proteins were mislocalized at low temperatures (Misu et al., 2003). Mislocalization of Bni1p-GFP and Gic1p-GFP was observed in >95 and >80% of the small-budded cdc50 mutant cells at 18°C, respectively. We next examined the localization of these proteins in the small-budded drs2 mutant. A C-terminally GFP-tagged allele that we generated was integrated into the genome as the sole source of BNI1 and GIC1. In the drs2 mutant grown at 18°C for 12 h, both Bni1p-GFP and Gic1p-GFP were mislocalized to punctate or tubular membranous structures, similar to those seen in the cdc50 mutant cells (Figure 1B). These Bni1p-GFP- and Gic1p-GFP-positive structures were often peripherally localized beneath the plasma membrane, but lacked polarized distributions. Mislocalization of Bni1p-GFP and Gic1p-GFP was seen in 68 and 50% of the small-budded drs2 mutant cells, respectively. These results suggest that Drs2p functions similarly to Cdc50p during polarized cell growth.

Cdc50p and Drs2p Are Localized to the TGN/Endosomal Membranes
We previously demonstrated that Cdc50p is localized to late endosomal/prevacuolar compartments (Misu et al., 2003). This study, however, also suggested that a fraction of Cdc50p may be localized to a compartment other than the late endosomal compartment. In contrast, Drs2p seemed to be colocalized with a TGN marker, Kex2p, by immunofluorescence microscopy (Hua et al., 2002). We examined the localization of Cdc50p to the TGN. To identify the TGN, we stained cells with another TGN marker, Sec7p (Franzusoff et al., 1991). We created diploid cells simultaneously expressing both a C-terminally GFP-tagged Cdc50p and a C-terminally mRFP1-tagged Sec7p to examine their distributions by fluorescence microscopy. Both Cdc50p-GFP and Sec7p-mRFP1 were observed as punctate structures scattered throughout the cell at 30°C (Figure 2A). Quantitative analysis of individual spots revealed that 57% of Cdc50p-GFP-positive structures were colocalized with Sec7p-mRFP1-positive structures (n = 315). Forty-eight percent of Sec7p-mRFP1-positive structures were colocalized with Cdc50p-GFP-positive structures (n = 371). We also introduced a C-terminally GFP-tagged Drs2p into diploid cells expressing Sec7p-mRFP1. Drs2p-GFP was colocalized with Sec7p-mRFP1 to a similar extent as that seen for the colocalization of Cdc50p with Sec7p (Figure 2A). Fifty-six percent of the Drs2p-GFP structures were colocalized with Sec7p-mRFP1 structures (n = 321). Conversely, 54% of Sec7p-mRFP1 structures were colocalized with Drs2p-GFP structures (n = 322), suggesting that both Cdc50p and Drs2p were localized to the TGN. To directly examine the colocalization of Cdc50p with Drs2p, we attempted to express an mRFP1-tagged Cdc50p; we failed, however, to detect Cdc50p-mRFP1 fluorescence, likely due to both the low fluorescence intensity of mRFP1 and the low expression levels for Cdc50p (our unpublished data).

View larger version (75K): [in this window] [in a new window]   Figure 2. Cdc50p and Drs2p are localized to the TGN and endosomal membranes. (A) Colocalization of Cdc50p-GFP and Drs2p-GFP with mRFP1-tagged Sec7 protein. Wild-type cells expressing either CDC50-GFP (YKT788) or DRS2-GFP (YKT789) and SEC7-mRFP1 were grown to early-mid logarithmic phase in YPDA medium at 30°C and observed after fixation in 1% formaldehyde. GFP- and mRFP1-tagged proteins were visualized using GFP (green) and G-2A (red) bandpass filters, respectively. Obtained images were merged to demonstrate the coincidence of the two signal patterns. Bar, 5 µm. (B) Accumulation of FM4-64 and Drs2p-GFP in the class E compartment of vps27 mutant cells. Cells expressing DRS2-GFP (YKT840) were grown to mid-late logarithmic phase in YPDA medium, labeled in 32 µM FM4-64 at 30°C for 15 min, and then chased in fresh medium at 30°C for 30 min. DIC images of the same cells were collected to visualize the vacuoles. FM4-64 and Drs2p-GFP images were acquired under the red and green fluorescence channels, respectively. A merged image of these two channels is also shown. Bar, 5 µm. (C) Localization of GFP-Snc1p in drs2 and cdc50 cells. pRS416 GFP-SNC1 was introduced into YKT39 (WT), YKT745 (drs2), YKT249 (cdc50), and YKT496 (lem3) strains. Cells were grown to early-mid logarithmic phase in SDA-Ura medium at 30°C and observed immediately by microscopy. GFP-tagged proteins were visualized using a GFP bandpass filter. Bar, 5 µm.

We next examined whether Drs2p is also localized to late endosomal/prevacuolar compartments in a manner similar to Cdc50p. To facilitate detection of late endosomal/prevacuolar compartments, we examined the vps27 mutant strain. In vps27 cells, recycling Golgi proteins and endosomal proteins that traffic as far as the late endosome accumulate in a late endosomal/prevacuolar compartment, termed the class E compartment (Piper et al., 1995). FM4-64, a fluorescent membrane marker for endocytosis, also accumulates in the class E compartment of vps27 cells (Vida and Emr, 1995). vps27 mutant cells expressing Drs2p-GFP were loaded with FM4-64 dye, followed by a 30-min chase in fresh medium at 30°C. Similar to Cdc50p-GFP (Misu et al., 2003), a fraction of Drs2p-GFP was localized to the class E compartment labeled by FM4-64 (Figure 2B). Sec7p-GFP was not localized to the class E compartment in vps27 mutant cells (our unpublished data). Thus, both Cdc50p and Drs2p were distributed between the late endosome and the TGN. Hua et al. (2002) reported that drs2 mutant cells exhibit mislocalization of Snc1p, an exocytic v-SNARE protein (Gerst, 1997) that cycles between the cell surface and internal structures (Lewis et al., 2000). The mislocalization of GFP-Snc1p in the drs2 mutant may be explained by a defect in the exocytosis of proteins from the Golgi to the plasma membrane (Hua et al., 2002). We examined the involvement of Cdc50p in the recycling of GFP-Snc1p. In wild-type cells, although there was some internal fluorescence, GFP-Snc1p was observed primarily at the plasma membrane, concentrated within buds and regions of polarized growth (Figure 2C). In the drs2 mutant cells at 30°C, the amount of GFP-Snc1p localized to the plasma membrane decreased concomitant with an increase in the staining of internal punctate structures, which may be either the early endosome or the TGN (Hua et al., 2002). cdc50 mutant cells at 30°C exhibited a more severe defect in the mislocalization of GFP-Snc1p. GFP-Snc1p at the plasma membrane was faint; significant staining was only observed in the punctate structures (Figure 2C). GFP-Snc1p was localized normally in lem3 mutant cells at 30°C. These results suggest that both Cdc50p and Drs2p are involved in the recycling of GFP-Snc1p, possibly acting at the exocytic transport of Snc1p from the TGN to the plasma membrane (Hua et al., 2002).

Coimmunoprecipitation of Cdc50p/Lem3p Family with Drs2p/Neo1p Family
Our evidence that cdc50 and drs2 mutants are phenotypically similar and that both Cdc50p and Drs2p are localized to the TGN and late endosome raises the possibility that Cdc50p physically associates with Drs2p. We therefore attempted to isolate a complex of these proteins by coimmunoprecipitation experiments. We previously described a C-terminally HA-tagged version of CDC50 that is functional (Misu et al., 2003). We also constructed and expressed a C-terminally Myc-tagged version of DRS2 in cells expressing Cdc50p-HA. Drs2p-Myc was immunoprecipitated from membrane protein extracts prepared by solubilization in 1% CHAPS. Under our immunoprecipitation conditions, a TGN marker Kex2p was not precipitated nonspecifically (our unpublished data), indicating that Drs2p-Myc was efficiently solubilized from membranes. The resulting immunoprecipitates were analyzed by immunoblot by using both anti-Myc and anti-HA antibodies. Cdc50p-HA was coimmunoprecipitated with Drs2p-Myc (Figure 3A). Cdc50p-HA could not be detected in either control immunoprecipitates by using a nonspecific IgG (our unpublished data) or immunoprecipitates from cells lacking DRS2-Myc (Figure 3A). In addition, immunoprecipitation of Cdc50p-HA with an anti-HA antibody specifically isolated Drs2p-Myc (our unpublished data). These results indicate that the association of Cdc50p-HA with Drs2p-Myc is specific. In several immunoprecipitation experiments, 10% of Drs2p-Myc was recovered from the lysate, and 10-20% of Cdc50p-HA in the lysate was coimmunoprecipitated (our unpublished data). Therefore, we conclude that Cdc50p and Drs2p form a stable complex. When the blot was probed with anti-Lem3p antibodies, a small amount of Lem3p (<3% in the lysate) also could be detected in the immunoprecipitates from Drs2p-Myc-expressing cells. These results also suggest that a small fraction of Drs2p-Myc may associate with Lem3p.

View larger version (26K): [in this window] [in a new window]   Figure 3. Coimmunoprecipitation of Cdc50p/Lem3p family members with Drs2p/Neo1p family members. 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 the MATERIALS AND METHODS. Myc-tagged P-type ATPases were immunoprecipitated with an anti-Myc antibody from membrane extracts. Immunoprecipitates were subjected to SDS-PAGE, followed by immunoblot analysis using antibodies against HA (top), Myc (middle), and Lem3p (bottom). Arrows indicate the bands of anti-Myc antibody polypeptides detected by secondary antibodies (anti-mouse IgG-HRP). The results shown are representatives of several experiments. The strains used were as follows: YKT755 (Drs2p-Myc Cdc50p-HA) and YKT283 (Cdc50p-HA) (A), YKT759 (Dnf1p-Myc Cdc50p-HA) and YKT283 (Cdc50p-HA) (B), and YKT761 (Neo1p-Myc Cdc50p-HA) and YKT283 (Cdc50p-HA) (C).

The interaction between Cdc50p and Drs2p prompted us to examine a possible interaction between Lem3p and Dnf1p, because 1) the lem3 or dnf1 mutation does not affect cell growth, but they show synthetic lethal interaction with cdc50 and drs2 mutations; and 2) both Lem3p and Dnf1p are localized to the plasma membrane (Hua et al., 2002; Kato et al., 2002; Hanson et al., 2003; Pomorski et al., 2003). We constructed and introduced a C-terminally Myc-tagged version of DNF1 into cells. Immunoprecipitation of Dnf1p-Myc with an anti-Myc antibody coimmunoprecipitated Lem3p (Figure 3B). In several immunoprecipitation experiments, 10% of Dnf1p-Myc was recovered from the lysate, and 20% of Lem3p in the lysate was coimmunoprecipitated (our unpublished data). Cdc50p-HA was not detectable in these immunoprecipitates, suggesting specificity of the interaction between Dnf1p and Lem3p. We immunoprecipitated Neo1p-Myc, which possesses a function unique among the members of the DRS2/NEO1 family. Neither Cdc50p-HA nor Lem3p was detectable in the immunoprecipitates (Figure 3C), suggesting that Neo1p does not associate with either Cdc50p or Lem3p. These data demonstrate that Cdc50p forms a complex with Drs2p, whereas Lem3p forms a complex with Dnf1p in vivo.

Association of Cdc50p with Drs2p Is Required for the ER Exit of the Cdc50p-Drs2p Complex
To elucidate the function of Cdc50p interaction with Drs2p, we examined the localization of Drs2p-GFP in the absence of Cdc50p by fluorescence microscopy. In the cdc50 mutant at 30°C, Drs2p-GFP occurred within typical endoplasmic reticulum (ER) structures in yeast, which morphologically envelop the nucleus and extend from perinuclear membranes that frequently reach the periphery of the cell proximal to the plasma membrane (Figure 4A). Costaining with 4,6-diamidino-2-phenylindole dye confirmed that the Drs2p-GFP-positive structures in the cdc50 mutant were perinuclear (our unpublished observation). These results suggest that Drs2p-GFP could not be transported out of the ER in the absence of Cdc50p. In the lem3 mutant at 30°C, Drs2p-GFP occurred within punctate structures scattered throughout the cell, as is seen in wild-type cells. We next examined whether the absence of Lem3p would have a similar effect on the localization of Dnf1p. A C-terminally GFP-tagged allele of DNF1 was generated and integrated into the yeast genome to be the sole source of DNF1. Dnf1p-GFP in wild type was found in internal membranes and small punctate structures underlying the plasma membrane, concentrated at sites of emerging buds, small buds, and the mother-daughter neck of dividing cells (Hua et al., 2002; Pomorski et al., 2003). Dnf1p-GFP occurred within typical ER structures in the lem3 mutant at 30°C but not in the cdc50 mutant (Figure 4A). In the absence of Cdc50p, the amount of Dnf1p-GFP localized to sites of polarization diminished in most cells even at 30°C, likely due to the defects in cell polarization resulting from the cdc50 mutation. The localization of Neo1p-GFP in the cdc50 mutant at 30°C did not differ from that seen in wild-type cells; Neo1p-GFP occurred in ER-like structures and in several peripheral punctate structures, some of which were associated with the ER (Figure 4A; Hua and Graham, 2003). These results indicate that Cdc50p and Lem3p are required for the ER exit of Drs2p and Dnf1p, respectively. In the drs2 mutant at 30°C, Cdc50p-GFP was also mislocalized (Figure 4B), suggesting that the stable association of Drs2p with Cdc50p within the ER is essential for the ER exit of the Cdc50p-Drs2p complex. To substantiate this conclusion, we examined the complex formation within the ER by using a temperature-sensitive sec12-4 mutant, in which the vesicle transport from the ER to the Golgi is blocked. Drs2p-Myc was immunoprecipitated in sec12-4 mutant incubated at 35°C for 1 h. Under these conditions, Cdc50p-GFP was confined within the ER (our unpublished observation). Cdc50p-HA was coimmunoprecipitated with Drs2p-Myc, and similarly, Lem3p was coimmunoprecipitated with Dnf1p-Myc (Figure 4C), indicating that the Drs2p-Cdc50p and Dnf1p-Lem3p complexes are formed within the ER.

View larger version (77K): [in this window] [in a new window]   Figure 4. Cdc50p-Drs2p and Lem3p-Dnf1p complex formation is required for the ER exit of these proteins. (A) Localization of Drs2p-GFP, Dnf1p-GFP, and Neo1p-GFP in wild-type, cdc50, and lem3 cells. (B) Localization of Cdc50p-GFP in wild-type and drs2 cells. Cells were grown to early-mid logarithmic phase in YPDA medium at 30°C and observed immediately by microscopy. GFP-tagged proteins were visualized using a GFP bandpass filter. The strains used were as follows: YKT768 (Drs2p-GFP in WT), YKT769 (Drs2p-GFP in cdc50), YKT770 (Drs2p-GFP in lem3), YKT771 (Dnf1p-GFP in WT), YKT772 (Dnf1p-GFP in cdc50), YKT773 (Dnf1p-GFP in lem3), YKT775 (Neo1p-GFP in WT), YKT776 (Neo1p-GFP in cdc50), YKT259 (Cdc50p-GFP in WT), YKT774 (Cdc50p-GFP in drs2). Bars, 5 µm. (C) Coimmunoprecipitation of Cdc50p and Lem3p with Drs2 and Dnf1p, respectively, in sec12-4 cells. sec12-4 cells were grown at 25°C to mid logarithmic phase in YPDA medium and then incubated at 35°C for 1 h. Drs2p-Myc or Dnf1p-Myc was immunoprecipitated with anti-Myc antibody, and the precipitated proteins were detected by immunoblot analysis as described for Figure 3. Arrows indicate anti-Myc antibody detected by secondary antibodies (anti-mouse IgG-HRP). The strains used were as follows: YKT913 (Drs2p-Myc Cdc50p-HA sec12-4), YKT914 (Dnf1p-Myc Cdc50p-HA sec12-4), and YKT915 (Cdc50p-HA sec12-4).

A C-terminally HA-tagged Lem3p was not fully functional, as cdc50 strains expressing LEM3-HA as a sole source of LEM3 exhibited a growth defect at 30°C, a permissive temperature for the parental cdc50 mutant (Figure 5A). Indirect immunofluorescence microscopy by using an anti-HA antibody revealed that Lem3p-HA was localized to internal membranes and small punctate structures underlying the plasma membrane concentrated at sites of polarized growth (Figure 5B), suggesting that Lem3p-HA transports normally out of the ER to the plasma membrane. Reinforcing this possibility, Dnf1p-GFP in LEM3-HA cells was localized properly to the plasma membrane at sites of polarization as observed in wild-type cells (Figure 5C). Lem3p-HA also was coimmunoprecipitated with Dnf1p-Myc (Figure 5D), indicating that Lem3p-HA forms a complex with Dnf1p-Myc within the ER, which is normally transported out of the ER. Lem3p likely plays an additional important role within the mature Lem3p-Dnf1p complex, after it has reached sites of polarization, which is independent from its structural role in the ER exit of the Lem3p-Dnf1p complex.

View larger version (70K): [in this window] [in a new window]   Figure 5. Function and localization of a C-terminally HA-tagged Lem3 protein. (A) Growth defect of cdc50 mutant strains expressing Lem3p-HA. A tetra type tetrad from a diploid cell, heterozygous for LEM3-HA and cdc50, was grown at 30°C. After streaking, YPDA plates were incubated at 30°C for 2 d. The result shown is representative of 10 independent tetrads. (B) Indirect immunofluorescence of Lem3p-HA. Cells expressing Lem3p-HA (YKT782) were prepared for immunofluorescence as described in MATERIALS AND METHODS. Cells were stained with an anti-HA antibody. Bar, 5 µm. (C) Localization of GFP-tagged Dnf1 protein in cells expressing Lem3p-HA. Cells expressing Dnf1p-GFP and Lem3p-HA (YKT809) were grown to early-mid logarithmic phase in YPDA medium at 30°C and observed immediately by microscopy. Bar, 5 µm. (D) Coimmunoprecipitation of Lem3p-HA with Dnf1p-Myc. Cells were grown at 25°C to a cell density of 0.5 OD600/ml in YPDA medium. Extracts prepared from YKT807 (Lem3p-HA Dnf1p-Myc) or YKT808 (Lem3p-HA) cells were subjected to immunoprecipitation with either an anti-Myc antibody or control mouse IgG. Immunoprecipitates were subjected to SDS-PAGE, followed by immunoblot analysis by using antibodies against HA (top) and Myc (bottom). An arrow indicates anti-Myc antibody detected by secondary antibodies (anti-mouse IgG-HRP).

Drs2p and Cdc50p Seem to Cycle between the TGN and Plasma Membrane
The distinct subcellular localizations of Cdc50p-Drs2p and Lem3p-Dnf1p raise the question of how these molecular complexes can perform redundant functions. Hua et al. (2002) proposed that if Dnf1p cycles between the exocytic and endocytic pathways, it could be a transient occupant of the TGN. We hypothesize that Cdc50p-Drs2p also may cycle between the TGN and plasma membrane, possibly substituting for the function of Lem3p-Dnf1p at the plasma membrane. We examined the localization of Drs2p-GFP in a mutant of VRP1, a protein required for proper organization of cortical actin patches and the internalization step during endocytosis (Munn et al., 1995). Drs2p-GFP, which was barely detectable at the plasma membrane in wild-type cells, could be seen at the plasma membrane in the vrp1 mutant cells at 25°C (Figure 6), indicating that Drs2p is transported to the plasma membrane. This increased staining at the cell surface occurred concomitant with a reduction in the intensity of Drs2p-GFP intracellular staining. Similar results were obtained for Cdc50p-GFP (Figure 6), suggesting that the Cdc50p-Drs2p complex recycles between the TGN and plasma membrane. To confirm that Dnf1p cycles between the plasma membrane and internal membranes underlying the plasma membrane, we examined the localization of Dnf1p-GFP in vrp1 mutant cells at 25°C. The majority of Dnf1p-GFP staining in vrp1 mutant cells was localized to the plasma membrane, becoming only barely detectable on smaller punctate structures, indicating that Dnf1p also cycles between those membranes (Figure 6). These results suggest that Drs2p and Dnf1p cycles between the plasma membrane and internal membranes, such as the endosome and TGN, so that either pair can substitute for the function of the other.

View larger version (54K): [in this window] [in a new window]   Figure 6. Localization of GFP-tagged Drs2, Cdc50, and Dnf1 proteins in vrp1 cells. Wild-type and vrp1 cells expressing either DRS2-GFP, CDC50-GFP, or DNF1-GFP were grown to early-mid logarithmic phase in YPDA medium at 25°C and observed immediately by microscopy. GFP-tagged proteins were visualized using a GFP bandpass filter. The strains used were as follows: YKT768 (Drs2p-GFP in WT), YKT777 (Drs2p-GFP in vrp1), YKT259 (Cdc50p-GFP in WT), YKT834 (Cdc50p-GFP in vrp1), YKT771 (Dnf1p-GFP in WT), YKT804 (Dnf1p-GFP in vrp1). Bar, 5 µm.

Drs2p May Regulate the Translocation of NBD-labeled Phosphatidylethanolamine and Phosphatidylserine
Whereas the drs2 mutant exhibits defects in APT activity at the plasma membrane (Tang et al., 1996; Gomes et al., 2000), this result remains under dispute (Siegmund et al., 1998; Marx et al., 1999). Because Drs2p was localized to the TGN, it is difficult to detect its APT activity at the plasma membrane in wild-type cells. Cdc50p-Drs2p becomes confined to the plasma membrane, however, when endocytosis is blocked by mutations in VRP1. Kato et al. (2002) and Hanson et al. (2003) have reported that the lem3 mutant has a defect in the transport of NBD-PE and -PC across the plasma membrane. Therefore, it remains possible that, in the lem3 vrp1 mutant, we may be able to detect lipid transport activity of Cdc50p-Drs2p accumulated to the plasma membrane. Drs2p-GFP and Cdc50p-GFP were localized to the plasma membrane in lem3 vrp1 mutant cells at 25°C (Figure 7A), although the intracellular punctate staining became more intense than that seen in vrp1 mutant cells (Figure 6). The plasma membrane localization of Drs2p-GFP and Cdc50p-GFP was distinctly evident when these cells were compared with the lem3 mutant (Figure 7A).

View larger version (62K): [in this window] [in a new window]   Figure 7. Localization of GFP-tagged Drs2 and Cdc50 proteins and the accumulation of NBD-labeled phospholipids in lem3 and lem3 vrp1 cells. (A) Localization of Cdc50p-GFP and Drs2p-GFP in lem3 and lem3 vrp1 cells. The lem3 (left column) and lem3 vrp1 (middle column) cells expressing either CDC50-GFP or DRS2-GFP were grown to early to mid logarithmic phase in YPDA medium at 25°C and observed immediately by microscopy. The lem3 vrp1 cells harboring both pKT1266 (YEplac181 CDC50-EGFP) and pKT1468 (YEplac195 DRS2) (right column) were grown to early-mid logarithmic phase in SD-Leu-Ura medium at 25°C and observed immediately by microscopy. GFP-tagged proteins were visualized using a GFP bandpass filter. For observation of each image, the same exposure and processing parameters were used. The strains used were as follows: YKT836 (Cdc50p-GFP in lem3), YKT770 (Drs2p-GFP in lem3), YKT805 (Cdc50p-GFP in lem3 vrp1), YKT806 (Drs2p-GFP in lem3 vrp1), and YKT843 (lem3 vrp1) harboring pKT1266 and pKT1468. Bar, 5 µm. (B) Percentage of accumulation of NBD-labeled phospholipids of the deletion mutants relative to wild-type cells. pKT1472 (YEplac195 DRS2 CDC50) was introduced into YKT839 (lem3 vrp1) strain. YEplac195 was introduced into YKT39 (WT), YKT496 (lem3), and YKT839 (lem3 vrp1) strains. Cells carrying the appropriate plasmids were grown to early-mid logarithmic phase in SD-Ura medium at 25°C and labeled with either NBD-PE, -PC, or -PS for 30 min at 25°C. The percentage of average accumulation for the deletion mutants relative to wild-type cell is presented with ±SD of seven independent experiments.

After cell labeling with either NBD-PE, -PC, or -PS, we determined the average cell-associated NBD fluorescence per cell by flow cytometry as described previously (Siegmund et al., 1998). Fluorescent lipid accumulation was measured for wild-type, lem3, and lem3 vrp1 cells grown at 25°C in SD medium. Calculation of the cell-associated fluorescence in the mutants, expressed as a percentage of that seen for wild-type cells (Figure 7B) revealed that the uptake of NBD-PE in the lem3 mutant decreased to 46% that of the wild-type. In the lem3 vrp1 mutant, NBD-PE uptake was restored to 92% that of the wild-type, suggesting that Cdc50p-Drs2p is also involved in NBD-PE internalization. The uptake of NBD-PC in the lem3 mutant decreased to 17% of that seen in the wild-type. In the lem3 vrp1 double mutant, NBD-PC internalization was minimally restored to levels 37% that of the wild-type, suggesting that Cdc50p-Drs2p is weakly involved in NBD-PC internalization. Interestingly, the uptake of NBD-PS in the lem3 mutant was increased to 158% that of the wild-type. In the lem3 vrp1 mutant, NBD-PS uptake increased to 283% that of the wild-type, suggesting the strong involvement of Cdc50p-Drs2p in NBD-PS internalization. These results suggest that Cdc50p-Drs2p possesses an APT activity; we cannot, however, exclude the possibility that additional proteins localized to the plasma membrane upon Vrp1p deficiency contribute to increases in the uptake of NBD-PE, -PC, and -PS in the lem3 vrp1 mutant. To confirm that Cdc50p-Drs2p is responsible for the increased uptake of NBD-labeled phospholipids in the lem3 vrp1 mutant, we attempted to construct both a lem3 vrp1 drs2 and a lem3 vrp1 cdc50 mutant. The vrp1 mutation results in synthetic lethality when combined with drs2 and cdc50 mutations (our unpublished data). We examined the uptake of NBD-labeled phospholipids in lem3 vrp1 cells overexpressing both DRS2 and CDC50. When Cdc50p-GFP and Drs2p were overexpressed in lem3 vrp1 mutant cells, stronger GFP signals were observed at the plasma membrane than in the parental strain (compare top middle panel with top right panel in Figure 7A). Over-expression of Cdc50p and Drs2p markedly enhanced NBD-PE and NBD-PS uptake to 130 and 343% of wild-type levels, respectively, and minimally increased the incorporation of NBD-PC to 49% of wild-type levels (Figure 7B). These results suggest that the Cdc50p-Drs2p complex possesses APT activity, which translocates aminophospholipids more efficiently than PC.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cdc50p and Lem3p Are Potential -Subunits of Phospholipid-translocating P-Type ATPases
In this work, we demonstrate that Cdc50p and Lem3p associate with members of APT subfamily of the P-type ATPases. The assembly of Drs2p with Cdc50p on the ER membrane is essential for the ER exit of the Cdc50p-Drs2p complex. Similar observations have been made for the vertebrate plasma membrane Na⁺, K⁺-ATPase, another P-type ATPase consisting of a catalytic -subunit and a noncatalytic -subunit. Assembly of the complex occurs in the ER; the - and -subunits are mutually dependent for transport out of the ER (Geering, 1990). Unassembled - and -subunits seem to be retained in the ER through the action of the ER quality control machinery (Beggah et al., 1996). They showed that unassembled -subunits associate with BiP, a chaperone in the ER, until assembly with -subunits occurs. Another type of quality control mechanism was reported for the ER retention of unassembled subunit (Fet3p) of a yeast iron transporter (Sato et al., 2004). In this case, unassembled Fet3p is transported out of the ER but retrieved from the Golgi through Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi. Similar mechanisms may operate to retain unassembled subunits of phospholipid translocating P-type ATPases in the ER in yeast.

The -subunit is likely required for the regulation of the Na⁺, K⁺-pump transport activity of the mature complex (Geering et al., 1996). Therefore, the Na⁺, K⁺-ATPase ER assembly process establishes not only the basic structural interactions between the subunits, required for maturation of the oligomeric proteins, but also distinct, functional interactions involved in the regulation of mature protein's functional properties. HA-tagged Lem3p was transported normally to the cell surface in a form complexed with Dnf1p. This LEM3-HA exhibited a weak synthetic growth defect with the cdc50 mutation, suggesting that Lem3p performs a function in the mature Lem3p-Dnf1p complex that cannot be performed by the HA-tagged form after reaching the plasma membrane. Lem3p also may be involved in the regulation or activation of the catalytic activity of Dnf1p, as occurs for the -subunit of Na⁺, K⁺-ATPase.

Because the Cdc50/Lem3 family of proteins are conserved throughout evolution (Misu et al., 2003), it would be interesting to investigate whether a Cdc50 family protein associates with a P-type ATPase in other organisms. A phospholipid-translocating ATPase, however, may not associate with a Cdc50p/Lem3p-related protein. Neo1p did not associate with either Cdc50p or Lem3p by coimmunoprecipitation. Neo1p may associate with Crf1p; this protein is dispensable