title: Defined sites of interaction between subunits E (Vma4p), C (Vma5p), and G (Vma10p) within the stator structure of the vacuolar H+-ATPase.
abstract: Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.
authors.affiliation: School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Richard P O
authors.initials: RP
authors.lastname: Jones
authors.forename: Lyndsey J
authors.initials: LJ
authors.lastname: Durose
authors.forename: John B C
authors.initials: JB
authors.lastname: Findlay
authors.forename: Michael A
authors.initials: MA
authors.lastname: Harrison
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D020409:Molecular Motor Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational / Q000235:genetics; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020409:Molecular Motor Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C479898:VMA3 protein, S cerevisiae; C447616:VMA5 protein, S cerevisiae; C500733:Vma4 protein, S cerevisiae; C485339:VMA10 protein, S cerevisiae; D025262:Vacuolar Proton-Translocating ATPases