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Vol. 19, Issue 4, 1783-1797, April 2008

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Protein Kinases Fpk1p and Fpk2p are Novel Regulators of Phospholipid Asymmetry

Kenzi Nakano^*, Takaharu Yamamoto, Takuma Kishimoto^*, Takehiro Noji^*, and Kazuma Tanaka

Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate Schools of *Medicine and Life Science, Sapporo 060-0815, Japan

Submitted July 9, 2007; Revised December 20, 2007; Accepted January 8, 2008
Monitoring Editor: Sandra Lemmon

	ABSTRACT

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Type 4 P-type ATPases (flippases) are implicated in the generation of phospholipid asymmetry in membranes by the inward translocation of phospholipids. In budding yeast, the DRS2/DNF family members Lem3p-Dnf1p/Dnf2p and Cdc50p-Drs2p are putative flippases that are localized, respectively, to the plasma membrane and endosomal/trans-Golgi network (TGN) compartments. Herein, we identified a protein kinase gene, FPK1, as a mutation that exhibited synthetic lethality with the cdc50 mutation. The kinase domain of Fpk1p exhibits high homology to plant phototropins and the fungus Neurospora crassa NRC-2, both of which have membrane-associated functions. Simultaneous disruption of FPK1 and its homolog FPK2 phenocopied the lem3/dnf1 dnf2 mutants, exhibiting the impaired NBD-labeled phospholipid uptake, defects in the early endosome-to-TGN pathway in the absence of CDC50, and hyperpolarized bud growth after exposure of phosphatidylethanolamine at the bud tip. The fpk1 fpk2 mutation did not affect the subcellular localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Further, the purified glutathione S-transferase (GST)-fused kinase domain of Fpk1p phosphorylated immunoprecipitated Dnf1p and Dnf2p to a greater extent than Drs2p. We propose that Fpk1p/Fpk2p are upstream activating protein kinases for Lem3p-Dnf1p/Dnf2p.

	INTRODUCTION

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Despite experimental uncertainties concerning the distribution of lipids in organelle membranes, it is widely accepted that eukaryotic cell membranes have asymmetric lipid distribution. This phospholipid asymmetry has been most well characterized 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 (Devaux, 1991; Zachowski, 1993; Pomorski et al., 2001). Phospholipid asymmetry is generated and maintained by ATP-driven lipid transporters or translocases. The type 4 subfamily of P-type ATPases (also called "flippases") is implicated in the translocation of phospholipids from the external to the cytosolic leaflet (Graham, 2004; Pomorski et al., 2004; Holthuis and Levine, 2005; Devaux et al., 2006; Daleke, 2007). Five members of this subfamily (Drs2p, Neo1p, Dnf1p, Dnf2p, and Dnf3p) are encoded in the genome of the yeast Saccharomyces cerevisiae (Catty et al., 1997).

Though no type 4 P-type ATPase has been shown to exhibit phospholipid translocase activity in reconstitution experiments with purified enzymes and chemically defined vesicles, accumulating evidence suggests that the yeast ATPases possess this activity in membranes. Dnf1p and Dnf2p are localized to the plasma membrane, and loss of Dnf1p and Dnf2p abolishes ATP-dependent transport of fluorescent 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). Chemical labeling of outer-leaflet phospholipids and staining with a PE-specific probe showed that PE is exposed on the outer leaflet in dnf1 dnf2 mutant cells (Pomorski et al., 2003; Iwamoto et al., 2004). Drs2p is localized to endosomes and the trans-Golgi network (TGN), and Drs2p-dependent NBD-phospholipid translocase activity has been detected in Golgi membranes (Natarajan et al., 2004) and post-Golgi secretory vesicles (Alder-Baerens et al., 2006). In such vesicles, Drs2p was required for the asymmetric arrangement of PE (Alder-Baerens et al., 2006).

Our recent results suggest that the Drs2p and Dnf1p/Dnf2p/Dnf3p proteins form complexes with members of the conserved Lem3p-Cdc50p family of membrane proteins. Cdc50p, Lem3p, and Crf1p were coimmunoprecipitated with Drs2p, Dnf1p and Dnf2p, and Dnf3p, respectively, and these proteins were required for the endoplasmic reticulum (ER) exit of the P-type ATPases (Saito et al., 2004; Furuta et al., 2007). Although we should await purification of the native proteins to conclude that these putative phospholipid translocases except for Neo1p are composed of noncatalytic and catalytic subunits, their heteromeric nature is reminiscent of the -β subunit composition of the well-characterized P-type ATPase, Na⁺,K⁺-ATPase (Kaplan, 2002). It is currently unknown whether the Cdc50p family possesses regulatory function after the complex has reached its destination (e.g., the plasma membrane for Lem3p-Dnf1p; Noji et al., 2006).

These putative heteromeric flippases are essential for cell growth, with Cdc50p-Drs2p, Lem3p-Dnf1p/Dnf2p, and Crf1p-Dnf3p playing major, intermediate, and minor roles, respectively (Hua et al., 2002; Saito et al., 2004). Recent studies suggest that Cdc50p-Drs2p and Lem3p-Dnf1p are not stably associated with their primary localization sites; both Cdc50p-Drs2p and Lem3p-Dnf1p are recycled from the plasma membrane through early endosomes to the TGN and back to the plasma membrane (Saito et al., 2004; Liu et al., 2007). This overlapping localization may underlie the functional redundancy between Cdc50p-Drs2p and Lem3p-Dnf1p/Dnf2p. Combined mutation of these genes compromises vesicle transport pathways, including endocytic internalization at low temperatures (dnf1 dnf2 drs2; Pomorski et al., 2003), vacuolar transport of alkaline phosphatase from the TGN (drs2 dnf1; Hua et al., 2002), and transport from early endosomes to the TGN (cdc50 lem3 crf1; Furuta et al., 2007).

One important approach toward understanding the physiological significance of phospholipid asymmetry would be identification of the regulatory pathways signaling to flippases and exploration of the upstream signals that they receive. We have recently shown that Cdc50p-Drs2p interacts with Rcy1p, a potential effector of the Rab family small GTPases Ypt31p/32p (Chen et al., 2005), and have proposed that the Ypt31p/32p-Rcy1p pathway regulates Cdc50p-Drs2p to promote the formation of transport vesicles destined for the TGN from early endosomes (Furuta et al., 2007). Because Lem3p-Dnf1p/Dnf2p are involved in the endocytic recycling pathway in conjunction with Cdc50p-Drs2p (Furuta et al., 2007), it might be expected that Lem3p-Dnf1p and Lem3p-Dnf2p would be regulated by the Ypt31p/32p-Rcy1p pathway. However, neither Dnf1p nor Dnf2p interacts with Rcy1p (Furuta et al., 2007), suggesting that Lem3p-Dnf1p/Dnf2p are regulated in a different manner.

In this study, we identify a protein kinase gene, FPK1, in our collection of mutations that exhibited synthetic lethality with cdc50 (Kishimoto et al., 2005). Our results suggest that Fpk1p and its homologue Fpk2p are upstream regulatory protein kinases for Lem3p-Dnf1p and Lem3p-Dnf2p.

	MATERIALS AND METHODS

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Media and Genetic Methods
Strains were cultured in YPDA rich medium (1% yeast extract [Difco Laboratories, 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). Synthetic complete medium (SC) was SD medium containing all required nutritional supplements. When appropriate, 0.5% casamino acids (Difco) were added to SD medium without uracil (SDA-Ura). For induction of the GAL1 promoter, 3% galactose and 0.2% sucrose were used as carbon sources instead of glucose (YPGA and SGA-Ura). Growth sensitivity to duramycin was examined on YPDA plates containing 5 µM duramycin (Sigma, St. Louis, MO). Standard genetic manipulations of yeast were performed as described previously (Guthrie and Fink, 1991).

Escherichia coli strains DH5 and XL1-Blue were used for construction and amplification of plasmids. The lithium acetate method was used to introduce plasmids into yeast cells (Elble, 1992; Gietz and Woods, 2002).

Strains and Plasmids
Yeast strains used in this study are listed in Table 1. Yeast strains carrying complete gene deletions (fpk1 and fpk2), green fluorescent protein (GFP)-tagged genes (KEX2, MYO2, DNF1, and DNF2), monomeric red fluorescent protein 1 (mRFP1)-tagged SEC7, three tandem repeats of the influenza virus hemagglutinin epitope (3HA)-tagged genes (DNF1 and DNF2), and 13 x myc-tagged genes (DNF1, DNF2, DNF3, DRS2, and NEO1) were constructed by PCR-based procedures as described (Longtine et al., 1998; Goldstein and McCusker, 1999). The GAL1 promoter-inducible C-terminal FPK1 fragment (amino acids 445-893) tagged with glutathione S-transferase (P_GAL1-GST-FPK1N) was similarly constructed. All strains constructed by PCR-based procedures were verified by colony-PCR amplification to confirm that the replacement had occurred at the expected locus.

Isolation of New Mutants Synthetically Lethal with the cdc50 Mutation

Microscopic Observations
Most GFP- or mRFP1-tagged proteins were observed in living cells, which were grown to early-midlogarithmic phase, harvested, and resuspended in SC medium. Cells were mounted on microslide glass and immediately observed. Localization of GFP-Tlg1p and mRFP1-Snc1p was examined in fixed cells. Fixation was performed by addition of a commercial 37% formaldehyde stock (Wako Pure Chemicals, Osaka, Japan) into the medium to a final concentration of 0.5%, followed by a 10-min incubation at 30°C. After fixation, cells were washed twice with phosphate-buffered saline (PBS) and examined.

Endosomal structures were visualized by brief labeling with the lipophilic styryl dye FM4-64 (Invitrogen, Carlsbad, CA). Cells were grown to early-logarithmic phase in YPDA medium at 30°C for 3 h. Four OD₆₀₀ units of cells were labeled with 32 µM FM4-64 in 100 µl of YPDA medium for 30 min on ice. Cells were harvested by centrifugation, resuspended in 200 µl of fresh YPDA medium, and incubated at 30°C for 1 min. Cells were washed twice with 100 µl of ice-cold SC medium and immediately observed using a G-2A filter set. The vacuole lumen was visualized using Cell Tracker Blue CMAC (Invitrogen) according to the manufacturer's protocol.

Cells were observed using a Nikon ECLIPSE E800 microscope (Nikon Instec, Tokyo, Japan) equipped with an HB-10103AF super high pressure mercury lamp and a 1.4 NA 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-coupled device camera (C4742–95-12NR; Hamamatsu Photonics, Hamamatsu, Japan) using AQUACOSMOS software (Hamamatsu Photonics). Observations are compiled from the examination of at least 100 cells.

Ultrastructural observation of cells by conventional electron microscopy was performed using the glutaraldehyde-osmium fixation technique according to procedures described previously (Sakane et al., 2006).

Staining of Phosphatidylethanolamine with Biotinylated Ro09-0198 Peptide
PE staining in the outer leaflet of the plasma membrane was performed with biotinylated Ro09-0198 peptide (Bio-Ro) as described (Iwamoto et al., 2004) with the following modifications. Bio-Ro was prepared essentially as described (Aoki et al., 1994). A 1-ml culture of cells (midlogarithmic phase, generally at a cell density of 0.4–0.6 OD₆₀₀/ml) was harvested, resuspended in 20 µl of YPDA containing 100 µM Bio-Ro (for wild type), 30 µM (for lem3), or 15 µM (for fpk1 fpk2), and incubated for 13 h (for wild type and fpk1 fpk2) or 15 min (for lem3) on ice. The cells were washed once with PBS and fixed with 5% formaldehyde in PBS for 1 h at room temperature. After two washes with spheroplast buffer (1.2 M sorbitol, 0.1 M potassium phosphate, pH 7.4), cells were resuspended in 100 µl of spheroplast buffer containing 100 µg/ml zymolyase 100T (Seikagaku Kogyo, Tokyo, Japan) and 2.2 mg/ml β-mercaptoethanol (Wako Pure Chemicals) and incubated for 10 min at 30°C. After two washes with spheroplast buffer, spheroplast cells were attached to poly-L-lysine–coated multiwell slides, fixed in methanol and acetone, and incubated in PBS containing 0.1% bovine serum albumin (Sigma) for 20 min at room temperature. To visualize Bio-Ro, cells were washed three times with PBS and incubated in PBS containing 5 µg/ml fluorescein streptavidin (Vector Laboratories, Burlingame, CA) for 1 h at room temperature. After five washes with PBS, cells were stained with 4' 6-diamidino-2-phenylindole (DAPI), suspended in 90% glycerol containing n-propyl gallate, and observed using an FITC (for fluorescein) bandpass or a UV-1A filter set.

Internalization of Fluorescence-labeled Phospholipids into Yeast Cells
Large unilamellar vesicles containing NBD-phospholipids were prepared as described (Saito et al., 2004). 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). Fluorescently labeled phospholipid internalization experiments were performed as described previously (Kato et al., 2002; Saito et al., 2004). Briefly, cells were grown to early-midlogarithmic phase in YPDA media at 30°C. After dilution to 0.35 OD₆₀₀/ml in SC medium, cells were shaken for 30 min at 30°C with vesicles containing 40% NBD-phospholipids and 60% DOPC at a final concentration of 50 µM. Cells were then suspended in SC 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 using the CellQuest software (BD Biosciences, San Jose, CA). NBD green fluorescence was plotted on a histogram to allow calculation of the mean fluorescence intensity.

Efflux of Phospholipids from Yeast Cells
Fluorescently labeled phospholipid efflux experiments were performed as described by Hanson and Nichols (2001). Cells were labeled with NBD-phospholipids for 60 min at 30°C as described above. To achieve equal levels of fluorescence in the strains to be compared, the concentration of an NBD-lipid for loading was adjusted according to the capability of each strain for the uptake of NBD-phospholipid. For the uptake of NBD-PE, the lem3 mutant was incubated at 50 µM, whereas the wild type and the fpk1 fpk2 mutant were incubated at 2.5 and 3 µM, respectively. For NBD-PS, the fpk1 fpk2 mutant was incubated at 50 µM, whereas the wild type and the lem3 mutant were incubated at 5 and 2 µM, respectively. After labeling, the cells were washed three times with ice-cold SC medium, resuspended in SC medium, and incubated at 30°C. At the given time points, cells were rapidly cooled in an ice bath and analyzed by flow cytometry as described above.

Antibodies
Mouse anti-HA (HA.11) mAb was purchased from BabCO (Richmond, CA). Mouse anti-myc (9E10) mAb was purchased from Sigma. Rabbit anti-Kex2p and anti-Pma1p polyclonal antibodies were gifts from S. Nothwehr (University of Missouri, Columbia, MO) and R. Serrano (Polytechnic University of Valencia, Valencia, Spain), respectively. Mouse anti-Pep12p mAb was a gift from Y. Ohsumi (National Institute for Basic Biology, Okazaki, Japan). For immunoblot analysis, these antibodies were used at the following dilution: anti-HA and anti-myc, 1:1000; anti-Pma1p, 1:5000; anti-Kex2p and anti-Pep12p, 1:2000. 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).

Sucrose Gradient Fractionation
Fractionation of subcellular organelles based on sedimentation through a sucrose step gradient (Antebi and Fink, 1992) was performed according to procedures described previously (Misu et al., 2003). All fractions were assayed for the relevant distribution of marker proteins by immunoblotting, which was performed as described previously (Misu et al., 2003). SDS-PAGE samples were heated at 37°C for 30 min before loading.

Expression and Purification of the GST-Fpk1p-kinase Domain (GST-Fpk1Np)
The KKT345 (P_GAL1-GST-FPK1N) strain and the pKT1700 (pKO10-GST-FPK1N) or pKT1702 [pKO10-GST-FPK1(K525R)N] plasmid were constructed to express the C-terminal kinase domain of Fpk1p fused to the C-terminus of GST under the control of the glucose-repressible GAL1-promoter. KKT345 cells or KKT268 (fpk1 fpk2) cells harboring pKT1700 or pKT1702 were grown at 30°C to early-logarithmic phase in YPDA (for KKT345) or SDA-U (for KKT268) medium and harvested by centrifugation. The collected cells were resuspended in 1.2 l of YPGA (for KKT345) or SGA-U (for KKT268) medium to a cell density of 0.5 OD₆₀₀/ml and incubated at 30°C for 12 h. The cells were harvested, washed twice with water, and resuspended in TESD buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, and 5 mM dithiothreitol) at a volume equal to that of the cell pellet. The cells were lysed with glass beads using Multi-beads shocker (Yasui Kikai Co, Osaka, Japan). After addition of 20 ml of TESD buffer, the cell lysate was centrifuged at 3000 rpm for 5 min at 4°C, and the resulting supernatant was centrifuged at 20,000 x g for 30 min at 4°C. Ammonium sulfate was slowly added to the supernatant, and the fraction that precipitated between 40 and 60% (wt/vol) salt saturation was recovered by centrifugation at 20,000 x g for 30 min at 4°C. The pellet was dissolved in 20 ml of TESD buffer and loaded onto a glutathione Sepharose 4B column (GE Healthcare, Uppsala, Sweden). The column was washed twice with five bed volumes of TESD buffer and eluted 10 times with one bed volume of TESD buffer containing 10 mM reduced glutathione (Wako Pure Chemicals). A portion of each eluate fraction was subjected to SDS-PAGE, followed by staining with SYPRO ORANGE (Molecular Probes, Eugene, OR). The amount of recovered GST-Fpk1Np was estimated by densitometry with a FLA3000 fluorescent image analyzer (Fuji Photo Film, Tokyo, Japan), using bovine serum albumin as a reference protein. The second eluate fraction, which provided the highest concentration and apparent homogeneity of GST-Fpk1Np (our unpublished results), was used in the phosphorylation assays.

Immunoprecipitation of P-type ATPases and Phosphorylation Assays with GST-Fpk1Np
Immunoprecipitation of P-type ATPases tagged with 13 x myc was performed as described previously (Saito et al., 2004) with the following modifications. Briefly, cells were grown at 30°C to a cell density of 0.5 OD₆₀₀/ml in YPDA medium. Cells collected from 300 ml (Dnf1p-13myc, Dnf2p-13myc, Drs2p-13myc, and Neo1p-13myc) or 1.2 l (Dnf3p-13myc) of culture were washed twice with ice-cold water and resuspended in 1 ml of lysis buffer (10 mM Tris-HCl, pH 8.0, 300 mM sorbitol, 100 mM NaCl, and 5 mM MgCl₂) containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride). The cells were lysed with glass beads using Multi-beads shocker. 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, pellets were solubilized in 0.45 ml of IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate [CHAPS]) containing 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 7.5 µg of anti-myc antibody for 1 h at 4°C. The samples were rotated with 30 µl of protein G-Sepharose 4 Fast Flow (GE Healthcare) for 1 h at 4°C. The protein G-Sepharose beads were pelleted, washed twice with IP buffer, and washed twice with kinase buffer (50 mM Tris-HCl, pH 7.6, 5 mM MgCl₂, 1 mM EDTA, and 1 mM EGTA). The beads were equally divided into three tubes. One was subjected to immunoblot analysis with anti-myc antibody (1:1000 dilution), and the others were used for kinase assays. Relative amounts of immunoprecipitated P-type ATPases were estimated by chemiluminescence with a FLA3000 fluorescence image analyzer (Fuji Photo Film).

For kinase assays, beads were suspended in 45 µl of kinase buffer with or without 100 nM purified GST-Fpk1Np. Reactions were started by adding 5 µl of 1 mM [-³²P]-ATP (2000 cpm/pmol; Perkin Elmer-Cetus, Norwalk, CT; 222 KBq/pmol), followed by incubation at 30°C for 10 min. Reactions were stopped by adding 16.7 µl of 4x SDS-PAGE sample buffer, followed by incubation at 37°C for 30 min. Samples were resolved by SDS-PAGE, and phosphorylated proteins were visualized and ³²P incorporation into each P-type ATPase was quantified with a FLA3000 fluorescent image analyzer (Fuji Photo Film).

	RESULTS

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A Mutation in a Novel Protein Kinase Gene FPK1 Exhibits a Synthetic Lethal Genetic Interaction with the cdc50 Mutation
To identify genes involved in CDC50 and DRS2 function, we previously screened for mutations that are synthetically lethal with cdc50, resulting in isolation of four mutants, each of which harbored an allele of erg3, rgp1, vps1, or srv2 (Kishimoto et al., 2005). We expanded this screen and isolated alleles of seven genes: ypt6, gsg1/trs85, pep8, ubp3, neo1, sec3, and ynr047w. Ypt6p is a Rab family small GTPase that promotes fusion of recycling endocytic vesicles with the TGN membrane, and Rgp1p is a GDP/GTP exchange factor (GEF) for Ypt6p (Siniossoglou et al., 2000; Siniossoglou and Pelham, 2001). The genetic interaction with ypt6 and rgp1 seems to reflect the involvement of Cdc50p-Drs2p in the endocytic recycling pathway (Hua et al., 2002; Saito et al., 2004; Kishimoto et al., 2005; Furuta et al., 2007). Gsg1p/Trs85p is a nonessential subunit of the transport protein complex (TRAPP) II, which is proposed to be a GEF for Ypt31/32p Rab family small GTPases (Morozova et al., 2006). Our recent results showing that the Cdc50p-Drs2p complex interacts with Rcy1p, an effector of Ypt31/32p (Furuta et al., 2007), suggest that TRAPP II is an upstream regulator of Cdc50p-Drs2p. Synthetic lethal interactions with drs2 were also reported for mutations in other subunits of the TRAPP II complex, TRS33 and KRE11 (Sciorra et al., 2005). Pep8p (Vps26p) is a subunit of the retromer, the sorting machinery required for retrieval from the late endosome to the TGN (Seaman et al., 1998; Reddy and Seaman, 2001). Null mutations of VPS29 and VPS35, encoding other retromer subunits, also exhibited synthetic lethality with cdc50 (our unpublished data), implicating Cdc50p in the late endosome-to-TGN pathway. NEO1 is the one essential gene among five putative flippases in yeast. Synthetic growth defects of drs2 and the neo1-1 temperature-sensitive allele have been reported (Hua and Graham, 2003). Genetic interactions with ubp3 and sec3 implicate Cdc50p in unknown functions; Ubp3p is a ubiquitin-specific protease that regulates transport between the ER and Golgi compartments (Cohen et al., 2003), and Sec3p is a spatial landmark that defines sites for polarized secretion (Finger and Novick, 1998). YNR047W encodes a novel protein kinase and the results reported herein suggest that Ynr047wp is a regulatory protein kinase for Lem3p-Dnf1p and Lem3p-Dnf2p. We named YNR047W FPK1 (flippase kinase 1). The synthetic growth defect of the cdc50 fpk1 mutants was shown in Figure 1A by tetrad dissection of a sporulated diploid heterozygous for cdc50 and fpk1.

View larger version (67K): [in this window] [in a new window]   Figure 1. Identification of fpk1 as a mutation synthetically lethal with the cdc50 mutation. (A) Tetrad dissection of the cdc50/CDC50 FPK1/fpk1 diploid. Tetrad genotypes (TT, tetratype; PD, parental ditype; and NPD, nonparental ditype) are indicated, and the identities of the double mutant are shown in parentheses. (B) Comparison of the amino acid sequences of Fpk1p, Fpk2p, NRC-2, and phot2 (phototropin-2). The kinase domains were initially aligned using the CLUSTAL W program and the alignment was then optimized by the BOXSHADE program. Black and gray boxes indicate identical and similar amino acids, respectively. The GenBank accession numbers for the given sequences are as follows: FPK1 (CAA96328.1), FPK2 (CAA42256.2), NRC-2 (N. crassa; EAU37081.1), and phototropin-2 (phot2, A. thaliana; AAC27293.2). (C) Growth of the conditional PGAL1-HA-CDC50 fpk1 fpk2 mutant. Cells were serially diluted and spotted onto plates containing galactose (YPGA) or glucose (YPDA), followed by incubation at 30°C for 2 d. Strains used were KKT70 (WT, wild type), KKT72 (fpk1), KKT266 (fpk2), KKT268 (fpk1 fpk2), KKT127 (PGAL1-CDC50), KKT117 (PGAL1-CDC50 fpk1), KKT331 (PGAL1-CDC50 fpk2), and KKT330 (PGAL1-CDC50 fpk1 fpk2). (D) Defective growth of the Cdc50p-depleted fpk1(K525R) kinase-negative mutant. KKT117 (PGAL1-HA-CDC50 fpk1) harboring YCplac111 (vector), YCplac111- FPK1 (pFPK1), or YCplac111-FPK1(K525R) [pFPK1(K525R)] were grown on a YPDA plate at 30°C for 2 d.

Single or double knockout of FPK1 and FPK2 did not affect cell growth at 18, 30, or 37°C (Figure 1C and our unpublished results). To perform phenotypic analyses of the cdc50 fpk1 mutant, we first constructed a conditional mutant in the fpk1 background in which CDC50 is expressed under the control of the glucose-repressible GAL1 promoter. As shown in Figure 1C, the P_GAL1-HA-CDC50 fpk1 mutant grew normally in galactose-containing medium (YPGA), but exhibited only residual growth in glucose-containing medium (YPDA) at 30°C. Deletion of FPK2 in this strain resulted in a more severe growth defect, whereas the Cdc50p-depleted fpk2 mutant grew normally (Figure 1C). In addition, the growth defect of the Cdc50p-depleted fpk1 mutant was suppressed by overexpression of FPK2 (our unpublished results). These results suggest that Fpk1p and Fpk2p are functionally redundant, but that Fpk1p plays a major role. The FPK1(K525R) allele carries an amino acid substitution in the conserved ATP-binding site that results in a kinase-negative mutant protein (Hanks et al., 1988). The Cdc50p-depleted FPK1(K525R) mutant also exhibited a growth defect (Figure 1D), suggesting that Fpk1p performs its Cdc50p-related function by phosphorylating target proteins.

The Cdc50-depleted fpk1 fpk2 Mutant Exhibits Defects in the Retrieval Pathway from Early Endosomes to the TGN
The lem3 mutation also exhibits synthetic lethality with the cdc50 and rcy1 mutations (Saito et al., 2004; Furuta et al., 2007; Supplementary Figure S1). Similarly, the fpk1 mutation exhibited synthetic lethality with the drs2 and rcy1 mutations (our unpublished results); Drs2p is a potential catalytic subunit of Cdc50p (Saito et al., 2004), and Rcy1p is a regulatory factor for the Cdc50p-Drs2p complex (Furuta et al., 2007). These results suggest that Fpk1p/Fpk2p may be functionally related to Lem3p-Dnf1p/Dnf2p. We have recently reported that a temperature-sensitive cdc50-ts lem3 crf1 mutant exhibits severe defects in the retrieval pathway from early endosomes to the TGN (Furuta et al., 2007). Thus, we examined this pathway in the Cdc50p-depleted fpk1 fpk2 mutant. Tlg1p is a target-SNARE that is recycled between the TGN and early endosomes; GFP-Tlg1p thus shows a punctate pattern reminiscent of endosomal/TGN membranes (Holthuis et al., 1998; Siniossoglou and Pelham, 2001). Snc1p is an exocytic vesicle-SNARE that is recycled from the plasma membrane via early endosomes to the TGN; GFP-Snc1p is primarily localized to the plasma membrane at polarized sites where exocytosis is actively occurring, such as buds (Lewis et al., 2000). Because Tlg1p and Snc1p are both recycled via the retrieval pathway from early endosomes to the TGN, these two proteins accumulate in the same aberrant intracellular structures when this pathway is blocked (Furuta et al., 2007).

Cells expressing mRFP1-Snc1p and GFP-Tlg1p were grown in glucose medium for 6 h. Under these conditions, Cdc50p-depleted cells (P_GAL1-HA-CDC50) did not exhibit intracellular accumulation of mRFP1-Snc1p, unlike cdc50 cells (Saito et al., 2004), probably because 6-h incubation was insufficient to completely deplete Cdc50p as described previously (Sakane et al., 2006). Both mRFP1-Snc1p and GFP-Tlg1p were normally localized in wild-type, lem3, fpk1 fpk2, and P_GAL1-HA-CDC50 cells (Figure 2A). In contrast, mRFP1-Snc1p and GFP-Tlg1p were colocalized in abnormal membranous structures in the Cdc50p-depleted fpk1 fpk2 mutant as well as in the Cdc50p-depleted lem3 mutant; 92% (n = 105) of the mRFP1-Snc1p–positive structures were also labeled with GFP-Tlg1p in the Cdc50p-depleted fpk1 fpk2 mutant (Figure 2A).

View larger version (37K): [in this window] [in a new window]   Figure 2. The Cdc50-depleted fpk1 fpk2 mutant is defective in vesicle transport from early endosomes to the TGN. (A) mRFP1-Snc1p and GFP-Tlg1p colocalized in abnormal intracellular structures in the Cdc50p-depleted fpk1 fpk2 mutant. Cells carrying pKT1566 (YEplac181-GFP-TLG1) and pKT1563 (pRS416-mRFP1-SNC1) were grown at 30°C for 6 h in YPDA medium, followed by microscopic examination after fixation with 0.5% formaldehyde. Strains are those described in the legend for Figure 1C, KKT102 (lem3), and KKT293 (PGAL1-CDC50 lem3). Images were merged to compare the two signal patterns. (B) GFP-Snc1p-pm localized to the plasma membrane in the Cdc50-depleted fpk1 fpk2 mutant. Cells harboring pRS416-GFP-SNC1 pm were grown and examined as described in A. (C) Kex2p-GFP mislocalized to the vacuole in the Cdc50p-depleted fpk1 fpk2 mutant. KKT58 (WT), KKT346 (lem3), KKT350 (fpk1 fpk2), KKT347 (PGAL1-CDC50), KKT348 (PGAL1-CDC50 lem3), and KKT349 (PGAL1-CDC50 fpk1 fpk2) cells expressing Kex2p-GFP were grown in YPDA medium at 30°C for 6 h, labeled with CellTracker Blue CMAC at 30°C for 30 min, and observed by fluorescence microscope. Bars, 5 µm.

Furuta et al. (2007) also reported the mislocalization of C-terminally GFP-tagged Kex2p (Kex2p-GFP) in the cdc50-ts lem3 crf1 mutant. Kex2p is a TGN resident furin-like protease that is localized due to constant retrieval from late and early endosomes (Brickner and Fuller, 1997; Lewis et al., 2000). When cycling between endosomes and the TGN is impaired, Kex2p mislocalizes to the vacuole (Wilcox et al., 1992; Spelbrink and Nothwehr, 1999). We examined the localization of Kex2p-GFP in the Cdc50p-depleted fpk1 fpk2 mutant and compared it with vacuoles visualized by staining with CellTracker Blue CMAC. In wild-type cells and control mutants, Kex2p-GFP exhibited a punctate pattern reminiscent of TGN membranes, whereas in the Cdc50p-depleted fpk1 fpk2 and Cdc50p-depleted lem3 mutants, Kex2p-GFP was primarily localized to vacuoles with concomitant decrease of the punctate localization (Figure 2C).

In the cdc50-11 lem3 crf1 mutant, double membrane structures with ring-, horseshoe-, or crescent-like morphology were accumulated (Furuta et al., 2007). Because Snc1p was localized to these structures by immunoelectron microscopy (Furuta et al., 2007), they seemed to represent the intracellular membranes visualized by light microscopy. We examined membranous structures that accumulated in the Cdc50p-depleted fpk1 fpk2 mutant by electron microscopy. When grown in YPDA medium at 30°C for 8 h to deplete Cdc50p, the P_GAL1-HA-CDC50 fpk1 fpk2 mutant cells accumulated double membrane structures (approximately nine structures [>200 nm in diameter]/10 µm²; n = 31 sections) to an extent similar to that in the P_GAL1-HA-CDC50 lem3 mutant (10 structures/10 µm², n = 35 sections; Figure 3). The P_GAL1-HA-CDC50 mutant cells accumulated smaller numbers of these structures (approximately four structures/10 µm², n = 34 sections; Figure 3). By light microscopy, however, the accumulation of these structures was not apparent (Figure 2A); this difference may be due to longer depletion of Cdc50p (8 h) in the cells used for electron microscopy than in those for light microscopy (6-h depletion). Abnormal membrane structures were not observed in wild-type, lem3, or fpk1 fpk2 cells. Taken together, the Cdc50p-depleted fpk1 fpk2 and Cdc50p-depleted lem3 mutants exhibited similar defects in the early endosome-to-TGN transport pathway.

View larger version (68K): [in this window] [in a new window]   Figure 3. Electron microscopic observation of Cdc50-depleted fpk1 fpk2 mutant cells. (A) KKT61 (WT), KKT102 (lem3), KKT268 (fpk1 fpk2), KKT287 (PGAL1-CDC50), KKT293 (PGAL1-CDC50 lem3), and KKT330 (PGAL1-CDC50 fpk1 fpk2) cells were cultured in YPDA at 30°C for 8 h. The cells were fixed with glutaraldehyde-osmium and processed for electron microscopic observation. Arrows and arrowheads indicate representative double-membrane structures with ring- and crescent-shaped morphology, respectively. Bar, 1 µm. (B) Quantitation of abnormal and large (>200 nm in diameter) double membranous structures. Bars represent the number of structures per 10 µm2. More than 30 cell sections were examined for each strain.

The fpk1 fpk2 Mutant Is Defective in the Inward Translocation of Phospholipids

We thus examined whether the fpk1 fpk2 mutations affect the inward translocation of phospholipids at the plasma membrane. If the fpk1 fpk2 mutant is defective in flipping phospholipids, a phospholipid enriched in the inner leaflet, such as PE, would be exposed on the outer leaflet of the plasma membrane. Ro09-0198 is a 19-amino acid tetracyclic polypeptide that forms a tight complex with PE in biological membranes (Choung et al., 1988). The lem3/ros3 and dnf1 dnf2 mutants exhibited hypersensitive growth to Ro09-0198 (Kato et al., 2002; Pomorski et al., 2003) and to duramycin, an analogue of Ro09-0198 (Noji et al., 2006). Wild-type, lem3, fpk1, fpk2, and fpk1 fpk2 strains were grown in YPDA medium containing 5 µM duramycin at 30°C. The fpk1 fpk2 mutant showed sensitivity to duramycin, but not to the same extent as the lem3 mutant (Figure 4A). The kinase-negative FPK1(K525R) fpk2 mutant also exhibited duramycin sensitivity (our unpublished results). In contrast, neither cdc50 nor drs2 mutant exhibited sensitivity to 5 µM duramycin (Supplementary Figure S2).

View larger version (32K): [in this window] [in a new window]   Figure 4. Phosphatidylethanolamine (PE) is exposed on the outer leaflet of the plasma membrane in the fpk1 fpk2 mutant. (A) Growth sensitivity of the fpk1 fpk2 mutant to duramycin. KKT70 (WT), KKT102 (lem3), KKT72 (fpk1), KKT266 (fpk2), and KKT268 (fpk1 fpk2) strains were cultured in YPDA medium at 30°C, serially diluted, and spotted onto a YPDA plate containing 5 µM duramycin, followed by a 1.5-d incubation at 30°C. (B) PE exposure at the polarized site during bud growth in the fpk1 fpk2 mutant. Wild-type (WT), lem3, and fpk1 fpk2 cells in A were grown to early-midlogarithmic phase, incubated in YPDA medium at 18°C for 3 h, and treated with 100, 30, or 15 µM biotinylated Ro09-0198, respectively, for 13 h (WT and fpk1 fpk2) or 15 min (lem3) on ice. After fixation, cells were spheroplasted and incubated with fluorescein streptavidin (Bio-Ro) and DAPI (to distinguish cell cycle stage) as described in Materials and Methods. Note that Bio-Ro staining in wild-type cells was observed only in the small-budded stage, whereas staining in lem3 and fpk1 fpk2 cells remained until the large-budded stage (arrows). (C) Localization of Myo2p-GFP in the fpk1 fpk2 mutant. KKT75 (WT), KKT351 (lem3), and KKT353 (fpk1 fpk2) strains expressing Myo2p-GFP were cultured as in B. Large-budded cells with divided nuclei, recognized by DAPI staining, were categorized as having Myo2p-GFP polarized to the bud tip (black), localized to the bud cortex (gray), and delocalized (white) (n > 100). The bud neck localization pattern, which was excluded from the categorization, was 53.8% (WT), 63.0% (lem3), and 49.3% (fpk1 fpk2) in late mitotic cells. A representative cell image is shown in the left panel. Bars, 5 µm.

Very recently, we have shown that the elongated bud morphology of the lem3 mutant is due to defects in the switch from apical to isotropic bud growth (apical-isotropic switch), which is activated by G2 cyclins-Cdc28p (Saito et al., 2007). Lem3p-Dnf1/2p-mediated translocation of phospholipids into the inner leaflet of the apical plasma membrane seems to activate GTPase-activating protein (GAP) activities of Rga1/2p toward the small GTPase Cdc42p, resulting in the dispersal of Cdc42p from the bud tip. In the lem3 mutant, polarity proteins including a type V myosin Myo2p remained polarized at the bud tip even at a late mitotic phase. We thus examined this phenotype in the fpk1 fpk2 mutant. The fpk1 fpk2 cells expressing Myo2p-GFP were grown asynchronously, and the localization of Myo2p-GFP was examined in late mitotic cells recognized by DAPI staining. In this analysis, cells with Myo2p-GFP at the bud neck were excluded, because the ratio of the bud neck localization pattern in late mitotic cells was not very different among the stains examined (see the legend to Figure 4C). As shown in Figure 4C, most of the fpk1 fpk2 and lem3 cells exhibited polarization of Myo2p-GFP to the bud tip (68.6 and 75.7%, respectively), whereas only 10.9% of the wild-type cells did. These results indicate that the fpk1 fpk2 mutant is also defective in the apical-isotropic growth switch.

We next examined whether the uptake of NBD-labeled phospholipids (NBD-PE, NBD-PC, and NBD-PS) across the plasma membrane was affected in our strains. As described previously, the lem3 mutant exhibited clear defects in the uptake of NBD-PE (34 ± 1% of wild type) and NBD-PC (16 ± 1% of wild type; Kato et al., 2002; Hanson et al., 2003). In contrast, the uptake of NBD-PS was minimally affected (84 ± 7% of wild type; Figure 5A; Kato et al., 2002; Hanson et al., 2003). Pomorski et al. (2003) reported that NBD-PS uptake in dnf1 dnf2 cells was 50% of uptake in wild-type cells; this was also mild compared with the pronounced defects in NBD-PE and -PC uptake. Internalization of NBD-phospholipids was not affected in the fpk1 or fpk2 strains, but uptake was significantly decreased in the fpk1 fpk2 double deletion strain (62 ± 3% for NBD-PE and 66 ± 5% for NBD-PC relative to wild type; Figure 5A). These results are consistent with weaker sensitivity of the fpk1 fpk2 mutant to duramycin. Surprisingly, the uptake of NBD-PS was much more impaired in the fpk1 fpk2 mutant (34 ± 4% of wild type) than in the lem3 mutant. These results raise the interesting possibility that the fpk1 fpk2 mutations affect multiple phospholipid translocases. We confirmed that expression of wild-type FPK1, but not kinase-negative FPK1(K525R), restored NBD-phospholipid internalization in the fpk1 fpk2 mutant (Figure 5A, bottom panel).

View larger version (35K): [in this window] [in a new window]   Figure 5. Defective inward translocation of NBD-labeled phospholipids in the fpk1 fpk2 mutant. (A) Uptake of NBD-labeled phospholipids. Cells were preincubated in SC medium for 3 h at 30°C and labeled with NBD-PE, -PC, or -PS for 30 min at 30°C, and internalized NBD-phospholipids were quantitated by flow cytometry as described in Materials and Methods. Strains used were as follows: top panel, KKT70 (WT), KKT102 (lem3), KKT72 (fpk1), KKT266 (fpk2), and KKT268 (fpk1 fpk2); bottom panel, the wild-type (WT) strain transformed with YCplac111 (vector) and the fpk1 fpk2 mutant transformed with YCplac111 (vector), YCplac111-FPK1 (pFPK1), or YCplac111-FPK1(K525R) [pFPK1(K525R)]. Data are presented as average percentage of accumulation relative to wild-type cells ± SD of three independent experiments. (B) Efflux of NBD-PE and NBD-PS was not affected in the fpk1 fpk2 mutant. Phospholipid efflux in wild-type (KKT70, ), lem3 (KKT102, ), and fpk1 fpk2 (KKT268, ) cells was measured at the indicated times as described in Materials and Methods. Relative values for the initial fluorescence in the lem3 mutant were 91.6 ± 1.3 and 90.5 ± 16.5% of the wild type for NBD-PE and -PS, respectively, and those in the fpk1 fpk2 mutant were 96.3 ± 12.4 and 91.2 ± 3.0%, respectively. Data are presented as means ± SD of at least three independent experiments. (C) Simultaneous overexpression of DNF1 and LEM3 suppressed the duramycin-sensitive growth of the fpk1 fpk2 mutant. fpk1 fpk2 cells (KKT268) harboring YEplac181 and YEplac195 (vector), YEplac181 and YEplac195-FPK1 (pFPK1), or YEplac181-LEM3 and YEplac195-DNF1 (pDNF1/LEM3) were tested for duramycin sensitivity as described in the legend of Figure 4A. (D) Simultaneous overexpression of DNF1 and LEM3 restored the uptake of NBD-labeled phospholipids in the fpk1 fpk2 mutant. Wild-type (WT) and fpk1 fpk2 cells harboring YEplac181 and YEplac195 (vector) or YEplac181-LEM3 and YEplac195-DNF1 (pDNF1/LEM3) were assayed for NBD-phospholipid internalization. Results are presented as described in A.

Taken together, these results suggest that the reduction in accumulation of NBD phospholipids in the fpk1 fpk2 cells is due to a defect in the inward transport of these lipids across the plasma membrane.

One plausible function of Fpk1p/Fpk2p would be the upstream regulation of Lem3p-Dnf1p/Dnf2p. If this were the case, we would expect suppression of the fpk1 fpk2 mutations by overexpression of LEM3 and DNF1. The fpk1 fpk2 mutant was cotransformed with two multicopy plasmids encoding either LEM3 or DNF1. Simultaneous overexpression of LEM3 and DNF1 suppressed the duramycin-sensitive growth of the fpk1 fpk2 mutant to an extent similar to that of expression of FPK1. The uptake of NBD-PE, -PC, and -PS was also increased in these cells (Figure 5, C and D). Overexpression of LEM3 and DNF1 also suppressed the growth defects observed in the Cdc50p-depleted fpk1 fpk2 mutant (our unpublished results). In contrast, overexpression of FPK1 did not suppress the duramycin sensitivity of the lem3 mutant (our unpublished results). These results are consistent with the hypothesis that Fpk1p/Fpk2p are upstream regulatory kinases of Lem3p-Dnf1p.

The Localization of Dnf1p and Dnf2p Is Unaltered in the fpk1 fpk2 Mutant
Defective inward phospholipid movement across the plasma membrane in the fpk1 fpk2 mutant could be due to altered expression or localization of Lem3p-Dnf1p or Lem3p-Dnf2p. Given that Lem3p-Dnf1p is recycled through the endocytic recycling pathway (Saito et al., 2004; Liu et al., 2007), Fpk1p/Fpk2p might regulate localization of Lem3p-Dnf1p/Dnf2p at endosomal compartments or the plasma membrane. To examine these possibilities, we used strains expressing C-terminal GFP- or HA-tagged Dnf1p and Dnf2p from their endogenous loci (Pomorski et al., 2003; Noji et al., 2006). The expression levels of Dnf1p-HA and Dnf2p-HA, as estimated by immunoblotting, were similar in wild-type and fpk1 fpk2 strains (our unpublished results).

Localization of Dnf1p-HA and Dnf2p-HA was examined by subcellular fractionation on sucrose density gradients (Figure 6A). In wild-type cells, a plasma membrane marker, Pma1p (a proton ATPase; Serrano et al., 1986; Bagnat et al., 2001), and a late endosome marker, Pep12p (t-SNARE; Becherer et al., 1996; Lewis et al., 2000), were recovered in high- and low-density fractions, respectively. Kex2p, which is localized to endosomal/TGN compartments (Brickner and Fuller, 1997; Lewis et al., 2000), peaked at an intermediate density. Dnf1p-HA peaked at a density similar to that of Kex2p and was distributed into higher densities at which Pma1p fractionated. These results suggest that Dnf1p-HA is primarily localized to endosomal/TGN compartments and is partially localized to the plasma membrane. In contrast, Dnf2p-HA cofractionated with Pma1p, suggesting that it is localized to the plasma membrane. As shown in Figure 6A, the fractionation profiles of Dnf1p-HA and Dnf2p-HA were similar in fpk1 fpk2 cells.

View larger version (34K): [in this window] [in a new window]   Figure 6. Localization of Dnf1p and Dnf2p is not affected by the fpk1 fpk2 mutations. (A) Sucrose gradient centrifugation analysis of Dnf1p-HA and Dnf2p-HA in fpk1 fpk2 cells. Cell lysates were prepared from wild-type (WT) and fpk1 fpk2 cells expressing either Dnf1p-HA or Dnf2p-HA and fractionated in 18–65% sucrose step density gradients as described in Materials and Methods. Equivalent volumes from each fraction were subjected to SDS-PAGE, and proteins were detected by immunoblotting. Strains used were KKT39 (DNF1-HA), KKT342 (DNF2-HA), KKT340 (fpk1 fpk2 DNF1-HA), and KKT344 (fpk1 fpk2 DNF2-HA). (B) Localization of Dnf1p-GFP and Dnf2p-GFP in the fpk1 fpk2 mutant. Wild-type (WT) and fpk1 fpk2 cells expressing either Dnf1p-GFP (left) or Dnf2p-GFP (right) were grown to early-midlogarithmic phase in YPDA medium at 30°C, followed by fluorescence microscopic observation. Strains used were KKT33 (DNF1-GFP), KKT334 (DNF2-GFP), KKT332 (fpk1 fpk2 DNF1-GFP), and KKT336 (fpk1 fpk2 DNF2-GFP). (C) Punctate structures of Dnf1p-GFP costained with FM4-64. KKT33 (wild type) cells expressing Dnf1p-GFP were incubated with FM4-64 at 30°C for 1 min, followed by fluorescence microscopic observation. (D) Localization of GFP-Fpk1p. GFP-Fpk1p was expressed by transforming cells with the pKT1634 plasmid (pRS416-PFPK1-GFP-FPK1). Cells were grown to early-midlogarithmic phase in SDA-U medium at 30°C, followed by fluorescence microscopic observation. Top, KKT72 (fpk1) cells expressing GFP-Fpk1p were labeled with FM4-64 at 30°C for 1 min, and colocalization of GFP-Fpk1p and FM4-64 was examined. Arrowhead indicates the localization of GFP-Fpk1p at the plasma membrane. Bottom, KKT62 (SEC7-mRFP1) cells expressing GFP-Fpk1p were examined for colocalization of GFP-Fpk1p with Sec7p-mRFP1. (E) Colocalization of Dnf1p-GFP and mRFP1-Fpk1p. KKT33 (DNF1-GFP) cells expressing mRFP1-Fpk1p were examined for colocalization of Dnf1p-GFP with mRFP1-Fpk1p. Cells were grown to early-midlogarithmic phase in SDA-U medium at 30°C, followed by fluorescence microscopic observation. mRFP1-Fpk1p was expressed by transforming cells with the pKT1638 plasmid (pRS416-PTPI1-mRFP1-FPK1). In C–E, images were merged to compare the two signal patterns. Bars, 5 µm.

Localization of N-terminal GFP-tagged Fpk1p was examined by fluorescence microscopy. GFP-FPK1 was fully functional, because the Cdc50p-depleted GFP-FPK1 fpk2 mutant grew normally (our unpublished results). GFP-Fpk1p fluorescence appeared to be distributed throughout the cytoplasm. GFP-Fpk1p was also observed in punctate structures reminiscent of endosomal/TGN compartments in 37.7% of the observed cells (n = 154). In fact, 67.5% of these structures stained with FM4-64 after brief incubation (n = 114) and 62.5% colocalized with the TGN marker Sec7p-mRFP1 (n = 112; Franzusoff et al., 1991; Figure 6D). Furthermore, 64.9% of the mRFP1-Fpk1p punctate structures colocalized with Dnf1p-GFP dots (n = 111; Figure 6E). GFP-Fpk1p was also observed in the plasma membrane, albeit at low frequency (<10% of observed cells; Figure 6D, arrowhead). These results suggest that Fpk1p colocalizes with Dnf1p and Dnf2p at early endosomal/TGN compartments and at the plasma membrane, consistent with the notion that Fpk1p may be a regulatory protein kinase for Dnf1p and Dnf2p.

Phosphorylation of Dnf1p and Dnf2p by GST-Fpk1p In Vitro
We next determined whether purified Fpk1p would phosphorylate immunoprecipitated Dnf1p or Dnf2p in vitro. For this purpose, we expressed the C-terminal fragment of Fpk1p (amino acid residues 445–893) fused to the C-terminus of GST (GST-Fpk1Np) in yeast. GST-Fpk1Np was functional, because P_GAL1-GST-FPK1N drs2 mutant cells grew normally in galactose-containing medium, but not in glucose medium (our unpublished results).

GST-Fpk1Np was purified from yeast cell lysates by glutathione-Sepharose affinity column to apparent homogeneity, as assessed by SDS-PAGE (our unpublished results). Because Fpk1p has been grouped into the S6 kinase family (Hunter and Plowman, 1997), we first assayed GST-Fpk1Np for phosphorylation of the S6 peptide (AKRRRLSSLRA). GST-Fpk1Np phosphorylated the S6 peptide in a dose- and time-dependent manner at 30°C (our unpublished results). We next carried out kinase reactions on immunoprecipitated myc-tagged Dnf1p. Phosphorylated proteins were fractionated by SDS-PAGE, followed by autoradiography. GST-Fpk1Np phosphorylated Dnf1p-myc in a dose-dependent manner (our unpublished results). Immunoblot analysis showed that the immunoprecipitates also contained Lem3p, as previously described (Saito et al., 2004); however, no phosphorylation of Lem3p was observed (our unpublished results), suggesting specific phosphorylation of Dnf1p-myc.

We next determined whether GST-Fpk1Np would phosphorylate Dnf2p, Dnf3p, Drs2p, and Neo1p. The myc-tagged ATPases, including Dnf1p-myc, were immunoprecipitated from cells lacking FPK1 and FPK2, to eliminate possible FPK1/2-dependent endogenous phosphorylation. As shown in Figure 7A, in addition to Dnf1p, some extent of phosphorylation was observed for Dnf2p, Dnf3p, and Drs2p, but not for Neo1p. The r