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Figure 3


Fig. 3. Mutations in the profilin-like region of yeast B subunit do not affect assembly of bafilomycin-sensitive ATPase activity. (A) A yeast strain lacking yeast B subunit (VMA2{Delta}; lanes 1 and 4) was transformed with Vma2p (lanes 2 and 5) or Vma2pArch and separated by SDS-PAGE, blotted, and probed with monoclonal antibodies against yeast A subunit (top panel) or yeast B subunit (bottom panel). Lanes 1–3 are whole-cell blots; lanes 4–6 were immunoprecipitated with anti-B subunit and probed with anti-A subunit. The heavy chain (HC) of the immunoprecipitating antibody was detected in lanes 4–6. This experiment was repeated three times, the subunit A bands and heavy chain bands were analyzed by densitometry and the ratio of subunit A:heavy chain was determined. For wild-type cells the ratio was 0.94±0.02, for the Vma2pArch-transformed cells the ratio was 0.95±0.03. This indicates that very similar amounts of subunit A were immunopreciptated with anti-B antibody from VMA2{Delta} yeast transformed with wild-type yeast B subunit or Vma2pArch. BL, blank. (B) Vacuolar membranes were prepared for assay for bafilomycin-sensitive ATPase activity as described in Materials and methods. Samples of the vacuolar membranes from VMA2{Delta} yeast (lane 1), and VMA2{Delta} yeast transformed with yeast B subunit (lane 2) or with Vma2pArch plasmids (lane 3) were subjected to SDS-PAGE and immunoblotted to demonstrate the levels of subunit A and B associated with membranes. (C) ATPase assays were performed on the remainder of the membranes. Activities are expressed relative to vacuolar membranes isolated from the VMA2{Delta} strain transformed with Vma2p, which was defined as 100% and had a specific activity of 1.11 µmol l–1 ATP min–1 mg–1 protein in this experiment. Vacuolar membranes from the null yeast were significantly different from those of null yeast transformed with Vma2p (Student's t-test, *P<0.05). The membranes from the yeast transformed with Vma2pArch were also significantly different from those of the null yeast, but not significantly different from those of the yeast transformed with Vma2p.





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