Involvement of a serin like protease in the S. cerevisiae H+‐ATPase activation mechanism.

CAMPETELLI A N; MONESTEROLO N E; AMAIDEN M R; PREVITALI G; CASALE C H

San Miguel de Tucuman

Congreso; Sociedad Argentina de investigación Bioquímica y Biología Molecular; 2009

SAIB

We have shown that tubulin, in its acetylated form, interacts with some 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 wh ben stimulated with glucose, indicating that this protease could be involved in the H+-ATPase activation mechanism. Even though some authors have already proposed a possible involvement of a protease in this activation mechanism, it is the first report where the protease involvement is confirmed and the enzyme is partially identified.