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Cysteine-mediated Cross-Linking Indicates That Subunit C of the V-ATPase Is in Close Proximity to Subunits E and G of the V1 Domain and Subunit a of the V0 Domain

Cysteine-mediated Cross-Linking Indicates That Subunit C of the V-ATPase Is in Close Proximity to Subunits E and G of the V1 Domain and Subunit a of the V0 Domain

Takao Inoue, Michael Forgac

J Biol Chem. 2005 Jul 29;280(30):27896-903.

PMID: 15951435

Abstract:

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP92944713	4-(N-Maleimido)benzophenone		92944-71-3	Price

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