Identification of fpk1Δ as a mutation synthetically lethal with the cdc50Δ mutation. (A) Tetrad dissection of the cdc50Δ/CDC50 FPK1/fpk1Δ 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 P_GAL1-HA-CDC50 fpk1Δ fpk2Δ 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 (fpk1Δ), KKT266 (fpk2Δ), KKT268 (fpk1Δ fpk2Δ), KKT127 (P_GAL1-CDC50), KKT117 (P_GAL1-CDC50 fpk1Δ), KKT331 (P_GAL1-CDC50 fpk2Δ), and KKT330 (P_GAL1-CDC50 fpk1Δ fpk2Δ). (D) Defective growth of the Cdc50p-depleted fpk1(K525R) kinase-negative mutant. KKT117 (P_GAL1-HA-CDC50 fpk1Δ) harboring YCplac111 (vector), YCplac111- FPK1 (pFPK1), or YCplac111-FPK1(K525R) [pFPK1(K525R)] were grown on a YPDA plate at 30°C for 2 d.

The Cdc50-depleted fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ 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 (lem3Δ), and KKT293 (P_GAL1-CDC50 lem3Δ). Images were merged to compare the two signal patterns. (B) GFP-Snc1p-pm localized to the plasma membrane in the Cdc50-depleted fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ mutant. KKT58 (WT), KKT346 (lem3Δ), KKT350 (fpk1Δ fpk2Δ), KKT347 (P_GAL1-CDC50), KKT348 (P_GAL1-CDC50 lem3Δ), and KKT349 (P_GAL1-CDC50 fpk1Δ fpk2Δ) 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.

Electron microscopic observation of Cdc50-depleted fpk1Δ fpk2Δ mutant cells. (A) KKT61 (WT), KKT102 (lem3Δ), KKT268 (fpk1Δ fpk2Δ), KKT287 (P_GAL1-CDC50), KKT293 (P_GAL1-CDC50 lem3Δ), and KKT330 (P_GAL1-CDC50 fpk1Δ fpk2Δ) 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.

Phosphatidylethanolamine (PE) is exposed on the outer leaflet of the plasma membrane in the fpk1Δ fpk2Δ mutant. (A) Growth sensitivity of the fpk1Δ fpk2Δ mutant to duramycin. KKT70 (WT), KKT102 (lem3Δ), KKT72 (fpk1Δ), KKT266 (fpk2Δ), and KKT268 (fpk1Δ fpk2Δ) 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 fpk1Δ fpk2Δ mutant. Wild-type (WT), lem3Δ, and fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ) or 15 min (lem3Δ) 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 lem3Δ and fpk1Δ fpk2Δ cells remained until the large-budded stage (arrows). (C) Localization of Myo2p-GFP in the fpk1Δ fpk2Δ mutant. KKT75 (WT), KKT351 (lem3Δ), and KKT353 (fpk1Δ fpk2Δ) 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% (lem3Δ), and 49.3% (fpk1Δ fpk2Δ) in late mitotic cells. A representative cell image is shown in the left panel. Bars, 5 μm.

Defective inward translocation of NBD-labeled phospholipids in the fpk1Δ fpk2Δ 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 (lem3Δ), KKT72 (fpk1Δ), KKT266 (fpk2Δ), and KKT268 (fpk1Δ fpk2Δ); bottom panel, the wild-type (WT) strain transformed with YCplac111 (vector) and the fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ mutant. Phospholipid efflux in wild-type (KKT70, •), lem3Δ (KKT102, ○), and fpk1Δ fpk2Δ (KKT268, □) cells was measured at the indicated times as described in Materials and Methods. Relative values for the initial fluorescence in the lem3Δ 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 fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ mutant. fpk1Δ fpk2Δ 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 fpk1Δ fpk2Δ mutant. Wild-type (WT) and fpk1Δ fpk2Δ 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.

Localization of Dnf1p and Dnf2p is not affected by the fpk1Δ fpk2Δ mutations. (A) Sucrose gradient centrifugation analysis of Dnf1p-HA and Dnf2p-HA in fpk1Δ fpk2Δ cells. Cell lysates were prepared from wild-type (WT) and fpk1Δ fpk2Δ 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 (fpk1Δ fpk2Δ DNF1-HA), and KKT344 (fpk1Δ fpk2Δ DNF2-HA). (B) Localization of Dnf1p-GFP and Dnf2p-GFP in the fpk1Δ fpk2Δ mutant. Wild-type (WT) and fpk1Δ fpk2Δ 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 (fpk1Δ fpk2Δ DNF1-GFP), and KKT336 (fpk1Δ fpk2Δ 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-P_FPK1-GFP-FPK1). Cells were grown to early-midlogarithmic phase in SDA-U medium at 30°C, followed by fluorescence microscopic observation. Top, KKT72 (fpk1Δ) 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-P_TPI1-mRFP1-FPK1). In C–E, images were merged to compare the two signal patterns. Bars, 5 μm.

Dnf1p and Dnf2p are phosphorylated by GST-Fpk1ΔNp in vitro. (A) Phosphorylation of P-type ATPases by GST-Fpk1ΔNp. Myc-tagged ATPases were immunoprecipitated from fpk1Δ fpk2Δ mutant cells and subjected to in vitro kinase assays, as described in Materials and Methods. The fpk1Δ fpk2Δ mutants used were as follows: YKT1363 (DNF1-myc), YKT1364 (DNF2-myc), YKT1365 (DNF3-myc), YKT1366 (DRS2-myc), and YKT1367 (NEO1-myc). The arrow indicates autophosphorylation of GST-Fpk1ΔNp. (B) Immunoblot of P-type ATPases for quantification. Fractions of the immunoprecipitates in A were subjected to SDS-PAGE, followed by immunoblot analysis using anti-myc antibody. (C) Relative ³²P incorporation into P-type ATPases. The extent of ³²P incorporation into P-type ATPases was quantified and normalized to the amount of P-type ATPases in immunoprecipitates. ³²P incorporation into P-type ATPases in A was measured by a FLA3000 fluorescent image analyzer, and the amounts of immunoprecipitated P-type ATPases in B were estimated by chemiluminescence with a fluorescent image analyzer. The average percentage of phosphorylation relative to Dnf1p is presented ± SD of three (Dnf2p, Dnf3p, and Neo1p) or six (Dnf1p and Drs2p) independent experiments. (D) GST-Fpk1(K525R)ΔNp does not phosphorylate either Dnf1p nor Drs2p. In vitro kinase assays on Dnf1p and Drs2p were performed as described in A with GST-Fpk1ΔNp or GST-Fpk1(K525R)ΔNp, or without GST-Fpk1ΔNp. In these experiments, an equal amount of Dnf1p and Drs2p could be used, as shown in SYPRO ORANGE staining of the SDS-PAGE gel (E, arrows).