Loss of Regulators of Vacuolar ATPase Function and Ceramide Synthesis Results in Multidrug Sensitivity in Schizosaccharomyces pombe

Multidrug- and heavy-metal-sensitive phenotypes of lac1Δ and rav1Δ mutants. (A) A lac1Δ but not a lag1Δ mutant is sensitive to a range of drugs. The strains were plated on YE containing bleomycin (0.5 μg/ml), cycloheximide (CHX; 7.5 μg/ml), fluconazole (20 μg/ml), and camptothecin (50 μg/ml). After a week, on the control plate, the lac1Δ mutant had grown to the same extent as the wild type (wt) had after 3 days. (B) Wild-type and rav1Δ, lac1Δ, and lag1Δ mutant cells were plated onto YE (control) plates and YE containing 200 mM CaCl₂. (C) Wild-type and rav1Δ and lac1Δ mutant cells were plated onto YE (control) and YE containing either 1 mM ZnSO₄ or 0.2 mM CdSO₄. (D) rav1Δ mutant cells were transformed with the empty vector, the rav1 or lag1 (grav1 or glag1) genomic clone, or a pREP1 vector containing the cDNA of rav1 or lag1 (clag1 or clac1). The cells were plated on minimal medium alone or on minimal medium containing 200 mM CaCl_2, doxorubicin (50 μg/ml), or fluconazole (20 μg/ml). (E) lac1Δ cells were transformed with the empty vector, pREP1, the pREP41 vector containing the cDNA of lag1, or pREP1lac1. The cells were plated on minimal medium alone or medium containing 100 mM CaCl₂. (F) Wild-type cells were transformed with the empty pREP1 vector, pREP1lag1, or pREP1lac1 and plated on minimal medium alone or on medium containing doxorubicin (50 μg/ml), fluconazole (20 μg/ml), bleomycin (0.5 μg/ml), or cycloheximide (15 μg/ml). Plates were incubated for 2 days at 30°C.

Mutations in rav1 and lac1 lead to increased accumulation of doxorubicin. (A) The strains were plated on YE with 50 μg/ml doxorubicin. (B) Log phase cells were grown in YE containing 50 μg/ml doxorubicin before imaging. The size bar represents 15 μm. Images taken with longer exposure times are shown for wild-type (wt) and lag1Δ mutant cells so that the outline of cells that have not taken up the drug can be seen. (C) The numbers of cells displaying a fluorescent signal in panel A were quantified and expressed as a percentage of the total number of cells counted. At least 200 cells were observed for each strain, and the average of the data from three independent experiments is shown with standard deviations. (D) Higher magnification of a lac1Δ mutant cell exposed to doxorubicin as in panel B. For this image, the cells had been exposed to doxorubicin and then resuspended in water to promote vacuolar fusion. This enabled clearer observation of the vacuoles so that the cytoplasmic accumulation of doxorubicin and exclusion of the drug from the vacuoles could be visualized.

S. pombe Rav1 is part of a RAVE-like complex. (A) SpRav1 binds to Skp1. The Rav1-13myc protein was immunoprecipitated (IP) from whole-cell extracts (WCE) with anti-myc antibodies. A nontagged strain (wt) was used as a control. The experiment was also carried out with a strain where the skp1 gene is fused to the hemagglutinin epitope under the control of the nmt1 promoter. This was performed in the presence or absence of thiamine (Thi) to vary the amount of Skp1 expressed. Extracts were analyzed with anti-Skp1 antibodies. The immunoprecipitated Rav1 protein was detected with anti-myc antibodies. (B) Protein sequence alignment of S. cerevisiae Rav2 with its putative homologues from C. glabrata, C. albicans, and S. pombe. The accession numbers of the latter three sequences, respectively, are as follows: GenBank, CAG59121.1; Candida DB, CA1654; GenBank, CAA20308.1. Alignment was performed with ClustalX. Residues identical in all four species are shaded in black; those conserved across two or three species are shaded in dark gray; conservative changes are shaded in light gray. The amino acid positions are indicated on the right. (C) SpRav2 (SPBC3H7.12) interacts with Skp1. The Rav2-13myc protein was immunoprecipitated from whole-cell extracts. A nontagged strain (wt) was used as a negative control, and rav1-13myc was used as a positive control. Skp1 was detected as in panel A. The immunoprecipitated Rav2 protein was detected with anti-myc antibodies. (D) The S. pombe rav2Δ mutant is partially sensitive to drugs and calcium. The concentrations used were 50 μg/ml doxorubicin and 100 mM CaCl₂.

Rav1 interacts with the V-ATPase and influences its function. (A) Rav1-13myc protein was purified, and coprecipitating proteins were identified by gel electrophoresis and silver staining in comparison to the immunoprecipitate from a nontagged control (wt). The arrow corresponds to the prominent band observed upon silver staining, which was identified as Vma2. (B) Loss of Rav1 results in vacuolar acidification defects. Wild-type and rav1Δ mutant cells were exposed to quinacrine and imaged by fluorescence microscopy. Equal exposure times are shown for both strains. The size bar represents 10 μm. (C) Epistasis analysis of the rav1Δ and vma1Δ mutants. Wild-type and vma1Δ, rav1Δ, and vma1Δrav1Δ mutant cells were plated on 20 μg/ml fluconazole, 100 mM CaCl₂, and 50 μg/ml doxorubicin. (D) Rav1 protein is localized throughout the cytoplasm. Indirect immunofluorescence analysis was carried out with either wild-type untagged or rav1-13myc mutant cells with anti-myc. The scale bar represents 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole.