V1-ATPase from the yeast Saccharomyces cerevisiae was purified via a FLAG affinity tag introduced into the N terminus of the G subunit. The preparation migrated as a single band in native gel electrophoresis and contained subunits ABCDEFGH (with subunit C present at substoichiometric amounts) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The initial specific Ca-ATPase activity was approximately 6 micromol/min/mg. The structure of the yeast V1-ATPase was studied by electron microscopy of negatively stained and frozen hydrated samples. A 25-A resolution three-dimensional model of the complex was calculated from two-dimensional projections by the angular reconstitution technique. The model shows six elongated densities arranged in pseudo-3-fold symmetry around a large central cavity. At the top of the molecule, various protrusions can be seen. At the bottom of the complex, two large masses are visible that are connected to the main body of the molecule. Comparison of the yeast V1 structure with the structure of the intact V1V0-ATPase from bovine brain clathrin-coated vesicles (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804-31810) indicates that the structure of the isolated V1 from yeast is very similar to the structure of the V1 domain in the intact V-ATPase complex.

• PMID: 12960158
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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Zhang Z, Charsky C, Kane PM, Wilkens S

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