Abstract
The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.
Highlights
The N terminus of the V-ATPase subunit a functions in intracellular targeting and regulation of assembly
We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast
We chose for our modeling studies the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast, because of the wealth of structural data about the V-ATPase complex from yeast [1, 2] and because of the ease of introducing point mutations into the yeast protein
Summary
The N terminus of the V-ATPase subunit a functions in intracellular targeting and regulation of assembly. The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. The C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. All three classes operate by a rotary mechanism in which ATP hydrolysis in the peripheral domain drives rotation of a central rotor domain (composed of both peripheral and integral subunits) relative to the remainder of the complex (termed the stator domain) [11, 12] This rotation in turn drives proton translocation through the integral domain. Unlike the F- and A-type ATPases, which function to synthesize ATP, the physiological function of the V-ATPases is to translocate protons
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