Topological Characterization of the c, c′, and c″ Subunits of the Vacuolar ATPase from the Yeast Saccharomyces cerevisiae

Abstract

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that acidifies intracellular organelles in eukaryotes. Similar to the F-type ATP synthase (FATPase), the V-ATPase is composed of two subcomplexes, V 1 and V 0 . Hydrolysis of ATP in the V 1 subcomplex is tightly coupled to proton translocation accomplished by the V 0 subcomplex, which is composed of five unique subunits (a, d, c, c , and c؆). Three of the subunits, subunit c (Vma3p), c (Vma11p), and c؆ (Vma16p), are small highly hydrophobic integral membrane proteins called "proteolipids" that share sequence similarity to the F-ATPase subunit c. Whereas subunit c from the F-ATPase spans the membrane bilayer twice, the V-ATPase proteolipids have been modeled to have at least four transmembrane-spanning helices. Limited proteolysis experiments with epitope-tagged copies of the proteolipids have revealed that the N and the C termini of c (Vma3p) and c (Vma11p) were in the lumen of the vacuole. Limited proteolysis of epitope-tagged c؆ (Vma16p) indicated that the N terminus is located on the cytoplasmic face of the vacuole, whereas the C terminus is located within the vacuole. Furthermore, a chimeric fusion between Vma16p and Vma3p, Vma16-Vma3p, was found to assemble into a fully functional V-ATPase complex, further supporting the conclusion that the C terminus of Vma16p resides within the lumen of the vacuole. These results indicate that subunits c and c have four transmembrane segments with their N and C termini in the lumen and that c؆ has five transmembrane segments, with the N terminus exposed to the cytosol and the C terminus lumenal.

The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles inParamecium

“…1). They show considerable amino acid identity with c-subunits of other organisms, the same organization (with four membranespanning helices) (Flannery et al, 2004) and are highly hydrophobic molecules (Kawasaki-Nishi et al, 2003). The Paramecium c-subunits show the features described in other organisms; for example, this includes the conserved Glu137 in the yeast proteolipid vma3p (Hirata et al, 1997), and the three important Thr32, Phe136 and Tyr143 residues involved in binding of the V-ATPase inhibitor bafilomycin identified in Neurospora crassa (Bowman and Bowman, 2002) (Fig.…”

“…This question cannot be answered by the GFP labeling of csubunits alone, because any possible plasmalemmal staining would be obscured by the strong subcortical signal. Recently, c-and c′-subunits in yeast were shown to expose their N-and C-termini to the lumen of vacuoles (Flannery et al, 2004). In our study, we tagged the c-subunits C-terminally with GFP (see Materials and Methods), expecting that GFP should be exposed either to the lumen of organelles or to the extracellular space, if localized in the plasma membrane.…”

Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells

“…First, we have tagged the proteolipid c subunit of the V-ATPase with a V5 epitope tag at the C terminus. The topology of the c subunit ensures that the V5 tag in this construct will be exposed on the luminal, or, for plasma membrane localized V-ATPases, the extracellular surface of the protein (33). An added benefit of epitope tagging subunit c is that it is present in multiple copies per V-ATPase, making it likely that the V5 tag is present in nearly every V-ATPase in the cell (7,34).…”

A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

“…Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with antibodies. Antibodies used Fluorescence microscopy: Yeast were stained with quinacrine as previously described (Flannery et al 2004). Briefly, cells were grown overnight in YEPD pH 5.0 plus adenine, and diluted to a cell density of 0.25 OD 600 /ml in YEPD.…”