Reference: Tomashek JJ, et al. (1997) Reconstitution in vitro of the V1 complex from the yeast vacuolar proton-translocating ATPase. Assembly recapitulates mechanism. J Biol Chem 272(26):16618-23

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Abstract


Oligomeric assembly is a fundamental aspect of many complex enzymes. Using our native gel technique for examining subcomplexes of the V-ATPase V1 sector, we have developed an in vitro reconstitution assay for assembly of this complex. Assembly of complex II, the soluble V1 complex observed in native gels, is dependent upon the presence of divalent cations and physiological temperatures. Assembly of soluble V1 can occur in a stepwise fashion from smaller subcomplexes found in some strains deleted for V-ATPase subunits. Specifically, V1 can be assembled directly from complex III (subunits E and G) with complex IV (subunits A, B, D, and F) without prior disassembly of complex IV. The formation of complex III in vivo is also shown to be essential and could not be achieved in vitro. Assembly from simpler precursors is possible and is enhanced by added ATP. Assembly can be blocked by N-ethylmaleimide in a Vma1p (subunit A)-specific manner. From these data, we extend our previous model to consider an assembly pathway whose steps reflect the catalytic mechanism of the Boyer binding-change model.

Reference Type
Journal Article | Research Support, U.S. Gov't, P.H.S.
Authors
Tomashek JJ, Garrison BS, Klionsky DJ
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