INVOLVEMENT OF A SERIN LIKE PROTEASE IN THE S. cerevisiae H+-ATPASE ACTIVATION MECHANISM

CAMPETELLI AN; MONESTEROLO NE; AMAIDEN MR; PREVITALI G; ARCE CA; CASALE CH

San Miguel de Tucuman

Congreso; SAIB - th 45 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2009

We have shown that tubulin, in its acetylated form, interacts withsome P-ATPases inhibiting their enzymatic activities. In S.cerevisiae, the plasma membrane H+-ATPase is inhibited by tubulin and, upon glucose addition, the ATPase is activated and the tubulin is dissociated. Recently, we have shown that tubulin not only is dissociated from the ATPase, but it is also degraded. Our objective was to search a proteolytic enzyme responsible of tubulin degradation upon glucose stimulation. The physicochemical characterization of tubulin interacting proteases was performed by several chromatographic approaches and by using specific protease inhibitors. Haploid knock out yeast strains loosing the ORFs corresponding to several proteases were used. Biochemical experiments showed that in S. cerevisiae there is a citosolic protease that interacts with tubulin, it has a MW of ~45 kDa, a pI of 8-9 and is inhibited by serine protease inhibitors. The strain YOR084, loosing a serin protease of 44 kDa and pI 8.4 is deficient in H+-ATPase activation, at least, by tubulin degradation when stimulated with glucose, indicating that this protease could be involved in the H+-ATPase activation mechanism. Even thoughsome authors have already proposed a protease in this activationmechanism, it is the first report where the protease involvement isconfirmed and the enzyme is partially identified.