Purification Of The RAVE Complex From Saccharomyces Cerevisiae

In eukaryotic cells, vacuolar proton-translocating ATPases (V-ATPases) are present inendomembrane system, such as vacuoles, endosomes, lysosomes, Golgi apparatus, clathrin-coated vesicles and the plasma membrane of some cells. They play a central role in acidifcationof these organelles and in many normal and disease processes. Therefore the studies of structure,mechanism and regulation of V-ATPases are very important. Many studies in the yeast V-ATPase were carried out, but until now the regulation of the V-ATPase is not completelyunderstood.RAVE (Regulator of the H+-ATPase of the Vacuolar and Endosomal membranes) is anessential factor of assembly and reversible disassembly of V-ATPases. RAVE complex has threesubunits, which are Rav1p, Rav2p and Skp1p. There are few studies on RAVE. It is veryimportant to study structure of RAVE complex to understand more about the regulation ofassembly and reassembly at V-ATPases.FLAG tag is a polypeptide protein tag that consists of eight amino acids (N-AspTyrLysAspAspAspAspLys-C). It can provide high purity protein purification and low abilityto impair tagged protein structure. In this study, RAVE complex was purified by affinitypurification with the FLAG tag fused to subunit Rav1p or Rav2p. Three recombinant strainswere achieved when FLAG tag was fused to subunit Rav1p or Rav2p. They are Saccharomycescerevisiase BY4747FLAG-RAV1with FLAG tag fused to N-terminus of Rav1p, S. cerevisiaeBY4742RAV1-FLAG with FLAG tag fused to C-terminus of Rav1p and S. cerevisiae BY4742RAV2-FLAG with FLAG tag fused to C-terminuse of Rav2p.Yeast cells were incubated in8L YEPD medium at30oC,200rpm (OD600nmaround3).Furthermore, harvested cells were broken by a French pressure cell disruptor at25,000p.s.i inTBSE (50mM Tris/Cl,150mM NaCl,1mM EDTA, pH7.4) with1mm PMSF. The cell lysatewas centrifuged at20,000xg for20minutes at4oC. Then, the supernatant, was loaded onto asmall column containing1ml of anti-FLAG M2gel. After washing anti-FLAG column withTBSE, the RAVE complex was eluted with TBSE containing100μg/ml FLAG peptides. Theresults showed RAVE complex purification from strain with FLAG tag fused at C-terminus ofRav2p is better than that from strain with FLAG tag fused at N-terminus of Rav1p or C-terminusof Rav1p. Maldi-TOF was carried out to detect proteins in eluted solution. The results shownthat RAVE complex was successfully purified from Saccharomyces cerevisiase BY4742RAV2-FLAG.