SITE-DIRECTED MUTAGENESIS OF THE YEAST V-ATPASE B SUBUNIT (VMA2P)

摘要: Abstract The B subunit of the vacuolar (H)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and possess significant sequence homology with α mitochondrial F-ATPase, which forms major portion noncatalytic sites contributes several residues catalytic this complex. Based upon recent x-ray structure F ATPase (Abrahams, J. P., Leslie, A. G., Lutter, R., Walker, E.(1994) Nature 370, 621-628), site-directed mutagenesis yeast VMA2 gene carried out a strain containing deletion gene. encodes V-ATPase (Vma2p). Mutations at two postulated be contributed by Vma2p site (R381S Y352S) resulted complete loss activity proton transport, former having partial effect on assembly. Interestingly, substitution Phe for Tyr-352 had only minor effects (15-30% inhibition), suggesting requirement an aromatic ring position. Alteration Tyr-370, is near adenine pocket sites, Arg, Phe, or Ser caused 30-50% inhibition transport activity, that not essential Finally, region corresponding P-loop (H180K, H180G, H180D, N181V) also inhibited approximately 30-50%. None mutations either putative nor any ability correctly fold assemble. significance these results function discussed.