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ヨコヤマ ケン
YOKOYAMA KEN
横山 謙 所属 京都産業大学 生命科学部 先端生命科学科 職種 教授 |
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| 言語種別 | 英語 |
| 発行・発表の年月 | 2006/12 |
| 形態種別 | 研究論文 |
| 査読 | 査読あり |
| 標題 | Reconstitution in vitro of V-1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V-1 formation |
| 執筆形態 | その他 |
| 掲載誌名 | JOURNAL OF BIOLOGICAL CHEMISTRY |
| 出版社・発行元 | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| 巻・号・頁 | 281(50),pp.38582-38591 |
| 著者・共著者 | Hiromi Imamura,Saeko Funamoto,Masasuke Yoshida,Ken Yokoyama |
| 概要 | 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 V-o complex, responsible for proton translocation. The V1 complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, 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 A(1)B(1) and A(3)B(3) 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 A(1)B(1) 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. |
| DOI | 10.1074/jbc.M608253200 |
| ISSN | 0021-9258 |
| PMID | 17050529 |