Domain characterization and interaction of the yeast vacuolar ATPase subunit C with the peripheral stator stalk subunits E and G

Abstract

The proton pumping activity of the eukaryotic vacuolar ATPase (V-ATPase) is regulated by a unique mechanism that involves reversible enzyme dissociation. In yeast, under conditions of nutrient depletion, the soluble catalytic V 1 sector disengages from the membrane integral Vo, and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V1 subunit, C, is released into the cytosol. The affinities of the other V1 and Vo subunits for subunit Care therefore of particular interest. The C subunit crystal structure shows that the subunit is elongated and dumbbell-shaped with two globular domains (Chead and Cfoot) separated by a flexible helical neck region (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1148-1152). We have recently shown that subunit C is bound in the V1-Vo interface where the subunit is in contact with two of the three peripheral stators (subunit EG heterodimers): one via Chead and one via Cfoot (Zhang, Z., Zheng, Y., Mazon, H., Milgrom, E., Kitagawa, N., Kish-Trier, E., Heck, A. J., Kane, P. M., and Wilkens, S. (2008) J. Biol. Chem. 283, 35983-35995). In vitro, however, subunit C binds only one EG heterodimer (Féthière, J., Venzke, D., Madden, D. R., and Böttcher, B. (2005) Biochemistry 44, 15906-15914), implying that EG has different affinities for the two domains of the C subunit. To determine which subunit C domain binds EG with high affinity, we have generated Chead and Cfoot and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGCinteraction is Chead. In addition, we provide evidence that the EGChead interaction greatly stabilizes EG heterodimer. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.