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J Biol Chem, Vol. 274, Issue 37, 26057-26064, September 10, 1999

Substrate- and Inhibitor-induced Conformational Changes in the Yeast V-ATPase Provide Evidence for Communication between the Catalytic and Proton-translocating Sectors

Carolina Landolt-Marticorena, Walter H. Kahr, Paul Zawarinski, Judy Correa, and Morris F. Manolson^§

From the  Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada and ^§ Faculty of Dentistry, University of Toronto, Toronto, Ontario, M5G 1G6, Canada

The vacuolar-type H⁺-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V₁) involved in ATP hydrolysis and a membrane-associated complex (V₀) mediating proton translocation across a lipid bilayer. To date, little is known about the mechanism by which these two functions are coupled. We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex. Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests. ATP·Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V₁ sector. ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p. Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V₀ sector of the V-ATPase. Although the presence of divalent cations had no impact on the V₁ sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced. The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector. Addition of bafilomycin A₁, a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit. These data suggest that structural alterations within the V₁ sector result in alterations within the V₀ sector and vice versa. Clearly, a structural link must exist to couple the two sectors. The 95-kDa subunit is ideally suited to fulfill this role. Hydropathy analysis suggests a bipartite structure, with the NH₂-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments. Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH₂-terminal domain of the 95-kDa subunit is localized toward the cytosol. This region therefore is ideally positioned to interact with components of the V₁ complex, potentially functioning as the elusive link between the two sectors of the V-ATPase.

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