Domain characterization and interaction of the yeast vacuolar ATPase subunit C with the peripheral stator stalk subunits E and G.
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 V(o), and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V(1) subunit, C, is released into the cytosol. The affinities of the other V(1) and V(o) subunits for subunit C are therefore of particular interest. The C subunit crystal structure shows that the subunit is elongated and dumbbell-shaped with two globular domains (C(head) and C(foot)) 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 V(1)-V(o) interface where the subunit is in contact with two of the three peripheral stators (subunit EG heterodimers): one via C(head) and one via C(foot) (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 C(head) and C(foot) and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C(head). In addition, we provide evidence that the EGC(head) interaction greatly stabilizes EG heterodimer.
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Rebecca A
RA
Oot
Stephan
S
Wilkens
D002151:Calorimetry / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000502:physiology*
D025262:Vacuolar Proton-Translocating ATPases