Reconstitution in Vitro of V1 Complex of Thermus thermophilus V-ATPase Revealed That ATP Binding to the A Subunit Is Crucial for V1 Formation*

Vacuolar-type H+-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V1 complex, responsible for ATP hydrolysis, and a membrane-embedded Vo complex, responsible for proton translocation. The V1 complex of Thermus thermophilus V-ATPase has the subunit composition of A3B3DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V1 complex by reconstituting the V1 complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V1 complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A1B1 and A3B3 subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V1 complex. Kinetic analysis of assembly of the A and B monomers into the A1B1 heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.

• Publication year

  2006

• Venue

  Journal of Biological Chemistry

• Publication date

  2006-12-15

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/jbc.M608253200PMID 17050529
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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