Purification Of The RAVE-V₁Complex From Saccharomyces Cerevisiae Via Protein Tagging

RAVE (The Regulator of the proton-translocating ATPase of the Vacuolar andEndosomal membranes) is an essential factor for the reversible assembly of vacuolarproton-translocating ATPases. In yeast cells, when glucose is exhausted, the V-ATPasedissociates into V1and V0and the RAVE forms a complex with V1in the cytoplasm. In thisthesis, using the affinity chromatography, we purified RAVE-V1through the FLAG tag addedto the C terminus of Rav1p or Rav2p.By homologous recombination, we constructed two strains with a FLAG tag at the Cterminus of Rav1p or Rav2p, named BY4742RAV1-FLAG and BY4742RAV2-FLAG,respectively. The recombinant strain was cultured at30°C and200rpm in8L YPD mediumfor18h (OD600about3.5). The cells were incubated in the lysis buffer and broken by highpressure at low temperature. The supernatant was loaded onto a1-mL anti-FLAG affinitycolumn and the FLAG polypeptide was used for the elution of the protein complex.The results showed that FLAG tagging on the C terminus of the Rav2p was moresuitable to isolate the RAVE-V1complex and Rav2p was more stable than Rav1p during theisolation. Protein bands on the SDS-PAGE gel were identified by mass spectrometry. It wasconfirmed the presence of Vma1p (subunit A), Vma2p (subunit B), Vma4p (subunit E) andVma8p (subunit D) from V1, in addition to the three subunits of RAVE in the complex.Moreover, the Leu1p was found interacting with the RAVE for the first time. These resultsprovide a basis for future3D structural studies of the RAVE-V1complex and are conducive tofurther elucidate the molecular mechanism of RAVE regulation V-ATP activity.