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Abstract: Vacuolar ATPases use the energy derived from ATP hydrolysis, catalyzed in the A3B3 sector of the V1 ATPase to pump protons via the membrane-embedded VO sector. The energy coupling between the two sectors occurs via the so-called central stalk, to which subunit F does belong. Here we present the first low resolution structure of recombinant subunit F (Vma7p) of a eukaryotic V-ATPase from Saccharomyces cerevisiae, analyzed by small angle X-ray scattering (SAXS). The protein is divided into a 5.5nm long egg-like shaped region, connected via a 1.5nm linker to a hook-like segment at one end. Circular dichroism spectroscopy revealed that subunit F comprises of 43% α-helix, 32% β-sheet and a 25% random coil arrangement. To determine the localization of the N- and C-termini in the protein, the C-terminal truncated form of F, F1–94 was produced and analyzed by SAXS. Comparison of the F1–94 shape with the one of subunit F showed the missing hook-like region in F1–94, supported by the decreased D max value of F1–94 (7.0nm), and indicating that the hook-like region consists of the C-terminal residues. The NMR solution structure of the C-terminal peptide, F90–116, was solved, displaying an α-helical region between residues 103 and 113. The F90–116 solution structure fitted well in the hook-like region of subunit F. Finally, the arrangement of subunit F within the V1 ATPase is discussed. [ABSTRACT FROM AUTHOR]

Abstract: Understanding the structural traits of subunit G is essential, as it is needed for V1VO assembly and function. Here solution NMR of the recombinant N- (G1–59) and C-terminal segment (G61–114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G1–59 only. The residues of G1–59 involved in d binding are Gly7 to Lys34. The structure of G1–59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G1–59 are flexible. The surface charge distribution of G1–59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G1–59–d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E18–38 of subunit E of yeast V-ATPase and the presently solved structure of G1–59, both proteins have been docked and binding epitopes have been analyzed. [Copyright &y& Elsevier]

Abstract: The phosphatidylinositol 3-kinase Vps34p of Candida albicans participates in protein transport and in virulence. In order to characterize the functional link between these two activities we searched for proteins interacting with C. albicans Vps34p and demonstrate physical interaction of Vps34p with the subunit of the vacuolar H+-ATPase Vma7p. The interaction initially observed in a yeast two-hybrid system was confirmed in vitro with recombinant proteins. Functional assays show that the Vps34p protein is necessary for vacuolar acidification and growth at alkaline pH. In addition, the vps34 null mutant of C. albicans shows defective autophagocytosis. The relevance of these functions for virulence of C. albicans is discussed. [Copyright &y& Elsevier]