Why is Asp-286 conserved in the V-ATPase? : an honors thesis (HONRS 499)

Abstract

V-ATPases are multi-subunit proton pumps found on the cellular membranes of most organisms from yeast (Saccharomyces cerviseae) to humans. They are composed of two main components capable of reversible disassembly: V1 and V0. VI is composed of eight different subunits and is the peripheral domain, whereas Vo is composed of five different subunits and is the integral domain. The long-term objective of this research is to determine the function of one of these subunits on the V. complex: subunit d, encoded by the VMA6 gene. Subunit d is of interest because it serves as the bridge between V I and Vo.Asp-286 of subunit d is highly conserved in mammals, insects and fungi, suggesting that it may be important for V-ATPase function. Using QuikChange site-directed mutagenesis by PCR, a mutation was introduced in subunit d. The mutation substituted aspartic acid on position 286 with alanine. We hypothesized that if Asp-286 has a structural role in subunit d, this change would modify the tertiary structure of subunit d and possibly alter the activity and assembly of the V-ATPase complex.