The Yeast Leu1p Participates In The Regulation Of V-ATPases By The RAVE Complex

Vacuolar ATPase, widely exists in eukaryotic endomembrane system, is an enzyme thatuses the energy generated by hydrolysis of ATP to transfer H+. The function of V-ATPase isvery important for maintaining the stability of intracellular environment and cells’ normalphysiological condition. RAVE (regulator of the H+-ATPase of the vacuolar and endosomalmembrane) complex consist of Rav1p, Rav2p and Skp1p, which is a key regulatory factor ofV-ATPase activity regulatory mechanism.In this study, we purified RAVE complex and its related proteins from Saccharomycescerevisiae BY4742FLAG-RAV2through the method of affinity chromatography. The fusionprotein FLAG-Rav2p was confirmed by western blot and identified by mass spectrum, andthe results showed that in addition to the three subunits Rav1p, Rav2p, Skp1p of RAVEcomplex, but also including the A (Vma1p), B (Vma2p), E (Vma4p) and D (Vma8p) subunitsof V-ATP enzyme. And what’s more, it was confirmed that the Leu1p (α-isopropylmalateisomerase) existed in the purified protein. For further study of the relationship between theLeu1p and RAVE complex, we constructed four Saccharomyces cerevisiae BY4742recombinant strains by fusion PCR and homologous recombination which are lost their rav1,rav2, vma2and leu1gene, respectively.Further study of recombinant strains’ tolerance ability of pH an CaCl2showed that thegrowth of leu1gene-deficient strain (α-isopropylmalate isomerase (Leu1p) missing) is similarto rav1and rav2mutants, but has significant difference with vma2defect type strain.According to these results, we concluded that the regulation of RAVE for V-ATPase activity islikely to be achieved by the conjunction with Leu1p. Our study provided an important basisfor investigating the relationship between RAVE and Leu1p, and also the regulatorymechanism of the V-ATPase.