ATP SYNTHASE COMPLEX - PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES
ATP SYNTHASE COMPLEX - PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES
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DOI：
10.1074/jbc.270.5.2053
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发表时间：
1995-02-03
影响因子：
4.8
通讯作者：
HATEFI, Y
中科院分区：
文献类型：
--
作者：
BELOGRUDOV, GI;TOMICH, JM;HATEFI, Y
线粒体ATP合成酶复合物的催化区F-1和膜区F-0通过45埃长的柄连接在一起。了解茎的组成和结构对研究F-0和F-1之间的构象能转移机制至关重要。本文报道了交联实验揭示的茎秆亚基之间以及与F-1和F-0亚基之间的近邻关系。交联制备的材料为牛心脏亚线粒体颗粒（SMP）和f1缺陷SMP。交联剂为3种不同化学特异性和不同交联长度的试剂，交联剂范围为0 ~ 10埃。通过凝胶电泳和亚基特异性抗体对α、β - γ、δ、OSCP、F-6、A6L、a（亚基6）、b、c和d进行免疫印迹，鉴定出交联产物。结果表明，这两个b亚基构成了OSCP、d和F-6相互独立结合的茎主干。亚基b、OSCP、d和F-6与α和/或β交联，但不与γ或δ交联。A6L的cooh末端位于膜外，与d交联，但不与任何其他茎或F-1亚基交联。未检测到亚基a和c与任何茎或F-1亚基交联。在f -1缺失的SMP中，出现交联的b + b和d + F-6二聚体，b与OSCP之间的交联程度大大降低。将F-1添加到F-1缺陷颗粒中似乎可以逆转这些变化。用胰蛋白酶处理缺乏f -1的颗粒可迅速水解OSCP和F-6，将b分裂成膜结合的18-、12-和8-9-kDa抗原片段，这些片段与d和/或彼此交联。胰蛋白酶也去除了A6L的cooh末端部分，但其余部分仍与亚基d交联。模型显示了茎亚基之间的近邻关系，以及与F-1近端（底部）和膜-基质界面附近的α和β亚基之间的近邻关系。
The catalytic sector, F-1, and the membrane sector, F-0, of the mitochondrial ATP synthase complex are joined together by a 45-Angstrom-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F-0 and F-1. This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits of F-1 and F-0, as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particles (SMP) and F-1-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-linking from zero to 10 Angstrom. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits alpha, beta gamma, delta, OSCP, F-6, A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F-6 are bound independent of one another. Subunits b, OSCP, d, and F-6 cross-linked to alpha and/or beta, but not to gamma or delta. The COOH-terminal half of A6L, which is extramembranous, crosslinked to d but not to any other stalk or F-1 subunit. No cross-links of subunits a and c with any stalk or F-1 subunits were detected. In F-1-deficient SMP, cross-linked b + b and d + F-6 dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. The addition of F-1 to F-1-deficient particles appeared to reverse these changes. Treatment of F-1-deficient particles with trypsin rapidly hydrolyzed away OSCP and F-6, fragmented b to membrane bound 18-, 12-, and 8-9-kDa antigenic fragments, which cross-linked to d and/or with one another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near neighbor relationships of the stalk subunits with one another and with the alpha and beta subunits at a level near the proximal end (bottom) of F-1 and at the membrane-matrix interface are presented.