Vma8p-GFP fusions can be functionally incorporated into V-ATPase, suggesting structural flexibility at the top of V1

Abstract

The vacuolar ATPase (V-ATPase) complex of yeast (Saccharomyces cerevisiae) is comprised of two sectors, V(1) (catalytic) and V(O) (proton transfer). The hexameric (A(3)B(3)) cylinder of V(1) has a central cavity that must accommodate at least part of the rotary stalk of V-ATPase, a key component of which is subunit D (Vma8p). Recent electron microscopy (EM) data for the prokaryote V-ATPase complex (Thermus thermophilus) suggest that subunit D penetrates deeply into the central cavity. The functional counterpart of subunit D in mitochondrial F(1)F(O)-ATP synthase, subunit γ, occupies almost the entire length of the central cavity. To test whether the structure of yeast Vma8p mirrors that of subunit γ, we probed the location of the C-terminus of Vma8p by attachment of a large protein adduct, green fluorescent protein (GFP). We found that truncated Vma8p proteins lacking up to 40 C-terminal residues fused to GFP can be incorporated into functional V-ATPase complexes, and are able to support cell growth under alkaline conditions. We conclude that large protein adducts can be accommodated at the top of the central cavity of V(1) without compromising V-ATPase function, arguing for structural flexibility of the V(1) sector.

Keywords: V-ATPase; Vma8p; yeast.

Figure 1

Growth characteristics of strains. Strains tested expressed Vma8p–GFP (C–D) fusion proteins, or truncated versions of Vma8p (E–G) and were compared to parental strain YRD15 (A) and a vma8 null strain V8N (B). All strains were grown overnight in liquid YEPD medium. Cell density was adjusted to 5 × 10⁶ cells/mL, then samples of each culture were serially diluted five-fold and 2 μL aliquots of each dilution dropped out onto YEPD plates buffered to pH 7.5. Plates were incubated at 28 °C for 3 days. Strains which had failed to grow after 3 days were incubated for a further 3 days.

Figure 2

Expression of Vma8p-GFP fusion proteins. Yeast cultures were grown in liquid YEPD medium buffered to pH 7.5, with the exception of V8ΔC47-GFP which was grown in YEPD medium buffered to pH 5.5. Protein lysates of cells were subjected to SDS-PAGE under reducing conditions. Gels were transferred to PVDF membrane and probed with antibodies against GFP (monoclonal) or Vma8p (polyclonal) as indicated below the membrane strips. Blots were developed with Vistra ECF substrate and visualized using a Wallac Storm chemifluorescence scanner. Rainbow^TM Marker protein size standards are shown at left. A control lane containing unfused GFP purified after expression in bacterial cells has been included at left.

Figure 3

The fluorescence signal from GFP localizes to the vacuolar membrane in strains expressing Vma8p-GFP fusion proteins. Samples of cells grown in liquid YEPD were taken during the exponential phase of growth and imaged using a Fluoview FV500 Confocal Laser Scanning Microscope (Olympus, Australia) for either DIC or fluorescence. Cells were treated with FM 4-64, a dye that specifically labels the outer leaflet of the vacuolar membrane, and the fluorescence signal imaged and overlayed with the fluorescence signal for GFP [33].

Figure 4

The fluorescence signal from quinacrine localizes to the vacuolar lumen in YRD15 (wild-type) strain as well as in strains V8-GFP (expressing the full length Vma8p protein fused at its C-terminus to GFP), V8ΔC47 and V8ΔC40-GFP, but not in vma8 null strain (V8N) and V8ΔC47-GFP.