Article Figures & Data
Figures
- Fig. 1.
Density selection for vma mutants. Wild-type yeast strain SF838-1Dα and two previously identified vmamutants (SF838-1Dα vma2Δ::LEU2 and SF838-1Dα vma3Δ::URA3) were cultured (separately) to log phase in YEPD, pH 5.0. Cells were then transferred to YEP containing 3% glycerol and 2% ethanol and incubated at 30°C for 16 h. Wild-type, vma2Δ, andvma3Δ cells were mixed together in the ratio 18:1:1, respectively, to give a total of 108 cells. The mixed culture was washed once with size selection buffer (0.67 g of yeast nitrogen base per liter, 0.25 M sorbitol, 10 mM Tris-HCl [pH 7.5]) and resuspended in 500 μl of the same buffer. Two hundred microliters (4 × 107 cells) was carefully layered on 10 ml of a 95% isosmotic Percoll solution and centrifuged at 30,000 ×g for 10 min. The percentage of wild-type cells (▾) andvma mutant cells (□) present in each fraction was evaluated by plating a sample from each fraction on YEPD, pH 5.0, and then replica plating the samples to appropriate selective media in order to identify the percentages of Ura+ and Leu+ colonies (representing the vma mutants). Fractions were collected as 1-ml aliquots, beginning from the top of the gradient. The density gradient is indicated by the dashed line. Relative density of each fraction was measured with a handheld refractometer.
- Fig. 2.
Growth phenotypes of wild-type and vma cells. Cells were streaked on the medium indicated and incubated at 30°C for 2 days. The vma45 strain is YOY69-1Ca, and thevma41 and vma43 strains represent Vma− spores derived from a single backcross of the original mutants. The wild-type strain is SF838-1Dα, and thevma12-1 strain represents a new allele of VMA12identified in the screen described here.
- Fig. 3.
(A) Restriction map of pMEY69-1 and various subclones. Restriction endonuclease sites are indicated. B, BamHI; C,ClaI; E, EcoRI; H, HindIII; Rv,EcoRV; S, SalI; X, XbaI. The fragments indicated were subcloned into pRS316 to give the plasmids indicated at the right and then tested for complementation of the Vma−growth phenotypes of YOY69-1Ca. The hatched box represents the complementing subclone, and arrows indicate the direction and extent of sequence determination. The KEX2 gene is shown as stippled box. (B) Disruption of the KEX2 gene. A 1,056-bpAgeI-HpaI fragment within the KEX2 ORF was replaced with a 2.2-kb fragment containing the LEU2gene.
- Fig. 4.
Multicopy MKC7 and YAP3 suppress the growth defects of kex2-Δ1 mutants at pH 7.0. SF838-5Aa kex2Δ mutant cells were transformed with pYO19 (wild-type KEX2 on a low-copy-number plasmid),MKC7 on a 2μm plasmid, YAP3 on a 2μm plasmid, or YEp24 (the 2μm plasmid with no insert). Transformants were initially identified by growth on unbuffered SD−ura (pH approximately 5.7) and then streaked to unbuffered SD−ura (left plate) and grown for 3 days at 30°C or to SD−ura buffered to pH 7.0 (right plate) and grown for 5 days at 30°C. Growth of kex2-Δ1 cells transformed with the following plasmids is shown (clockwise from top): pYO19, 2μm-MKC7, 2μm-YAP3, and YEp24.
- Fig. 5.
Loss of vacuolar acidification in kex2mutants. Vacuolar acidification was assessed by quinacrine accumulation in the vacuole as described in Materials and Methods. Log-phase yeast cells were incubated in 500 μl of PBS, pH 7, containing 200 μM quinacrine for 5 min at 30°C. After being stained, cells were washed with 500 μl of PBS and resuspended in 100 μl of the same buffer. Cells were viewed with differential interference contrast optics for observation of normal cell morphology and by fluorescence microscopy with a fluorescein isothiocyanate filter for observation of vacuolar staining with quinacrine. Each monograph is a composite of three to four fields. The following strains were used: SF838-5Aa(wild type), SF838-5Aa kex2-Δ1 (kex2Δ), and YOY69-1Ca ( vma45).
- Fig. 6.
Assembly of V-ATPase in wild-type and kex2Δ mutant cells. Nondenaturing immunoprecipitation of V-ATPase from biosynthetically labeled yeast cells was performed as described previously (34). Monoclonal antibody 13D11 against the 60-kDa peripheral V1 subunit was used for immunoprecipitation. Immunoprecipitated proteins were separated by SDS-PAGE and visualized by autoradiography. The positions of previously identified subunits of V-ATPase are indicated. The arrow indicates the 38-kDa band that is present in kex2Δ mutant strains but not in wild-type strains. Positions of protein molecular mass standards are indicated on the left. The strains used are the same as those used in the experiment shown in Fig. 5. (A) Steps in V-ATPase assembly (5-min pulse, varied chase times); (B) final assembled V-ATPase complex (60-min pulse, 0-min chase).
- Fig. 7.
V-ATPase isolated from kex2-Δ1 vacuolar membranes is indistinguishable from that isolated from the wild type. (A) Vacuolar membrane vesicles were incubated in cracking buffer (50 mM Tris-HCl [pH 6.8], 8 M Urea, 1 mM EDTA, 5% SDS, 5% β-mercaptoethanol) for 20 min at 50°C for the 100-kDa V0 subunit or 70°C for the remaining subunits. Fifteen micrograms (for detection of the 100-kDa subunit) or 3 μg (for detection of all other subunits) of vacuolar protein was loaded in each lane. Proteins were detected on Western blots with alkaline phosphatase-conjugated antibodies. (B) V-ATPase was purified from vacuolar membrane vesicles as described in Materials and Methods. Proteins in fractions with peak ATPase activities were separated on an SDS–10% polyacrylamide gel and visualized by silver staining. Positions of known V-ATPase subunits are indicated on the right. The asterisk indicates the position of an unidentified protein that consistently copurifies with V-ATPase activity. The positions of protein molecular weight standards (in thousands) are indicated on the left.
Tables
- Table 1.
Yeast strains and genotypes
Strain name Description Reference or source SF838-5A MATα ura3-52 leu2-3,112 his4-519 ade6 60 SF838-1D MATα ura3-52 leu2-3,112 his4-519 ade6 pep4-3 60 SF838-1D vma2Δ MATαura3-52 leu2-3,112 his4-519 ade6 vma2Δ::LEU2 69 SF838-1D vma3Δ MATα ura3-52 leu2-3,112 his4-519 ade6 vma3Δ::URA3 29 CJRY20-3B MATα ura3-52 leu2-3,112 his3-Δ300 ade2-101 lys2-801 53 CJRY20-4B MAT a ura3-52 leu2-3,112 his3-Δ300 ade2-101 lys2-801 53 MEY14 MATα ura3-52 leu2-3,112 his4-519 ade6 pep4-3 vma41-1 This study MEY32 MATα ura3-52 leu2-3,112 his4-519 ade6 pep4-3 vma43-1 This study MEY69 MATa ura3-52 leu2-3,112 his4-519 ade6 pep4-3 vma45-1 This study YOY69-1Aα MATαura3-52 leu2-3,112 HIS3 HIS4 ade2 ade6 pep4-3 vma45-1 This study YOY69-1Ca MATa ura3-52 leu2-3,112 his3-Δ300 HIS4 ade6 pep4-3 vma45-1 This study SF838-5Akex2-Δ1 MATa ura3-52 leu2-3,112 his4-519 ade6 kex2Δ::LEU2 This study YOY69 MEY69 X CJRY20-3Bα This study YOY11 SF838-5Aa kex2Δ X MEY69-1Aα/pYO19 This study - Table 2.
Overlap of known kex2 andvma phenotypes
Testa Temp (°C) Test resultb with: Wild-type cells vma3Δ cells kex2Δ cells vma45-1 cells Growth on: YEPD 30 +++ + + + YEPD, pH 5.0 30 +++ ++ ++ ++ YEPD, pH 7.5 30 +++ −− −− −− YEPD + 4 mM Zn2+ 30 +++ −− −− −− YEP-glycerol 30 +++ −− −− −− YEPD 17 +++ −− −− −− YEPD, pH 5.0 17 +++ ++ ++ ++ YEPD, pH 7.5 17 +++ −− −− −− Vacuole staining with quinacrine 25 +++ −− −− −− - Table 3.
V-ATPase and proton pumping activities of vacuolar membrane vesicles isolated from wild-type andkex2-Δ1 cells
Strain V-ATPase activity (μmol/min/mg of protein)a % of wild-type activity Proton pumping (relative level of quenching Rate in first 15 s (min−1) Extent after 10 min SF838-5A (wild type) 3.0 ± 0.2 (3) 100 7.2 2.1 SF838-5Akex2-Δ1 3.1 ± 0.5 (3) 103 7.3 2.4 ↵a Vacuolar membrane vesicles were prepared as described in Materials and Methods, and the ATPase activity that is sensitive to 100 nM concanamycin A was determined. Activities are presented as means ± standard deviations (with the number of samples in parentheses).
b Levels of ATP-dependent proton pumping were determined by monitoring quinacrine fluorescence quenching. Activity is expressed as (arbitrary) fluorescence units/mg of protein, normalized to total fluorescence as described in Materials and Methods.
