THE C-TERMINAL POSITIVELY CHARGED REGION OF SUBUNIT-8 OF YEAST MITOCHONDRIAL ATP SYNTHASE IS REQUIRED FOR EFFICIENT ASSEMBLY OF THIS SUBUNIT INTO THE MEMBRANE F0 SECTOR
THE C-TERMINAL POSITIVELY CHARGED REGION OF SUBUNIT-8 OF YEAST MITOCHONDRIAL ATP SYNTHASE IS REQUIRED FOR EFFICIENT ASSEMBLY OF THIS SUBUNIT INTO THE MEMBRANE F0 SECTOR
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DOI：
10.1111/j.1432-1033.1991.tb16110.x
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发表时间：
1991-07-01
期刊：
影响因子：
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通讯作者：
NAGLEY, P
中科院分区：
文献类型：
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作者：
GRASSO, DG;NERO, D;NAGLEY, P
本文研究了酵母线粒体ATP合成酶亚基8的截断衍生物，其中一个保守的带正电的残基（Lys47）和c端残基（Leu48）被定点诱变去除。该衍生物已被表达为嵌合前体N9L/Y8-1(K47-STP)，携带n端可切割先导序列（N9L），通过短桥接序列与截断的亚基-8客运蛋白融合。N9L/Y8-1 （K47-STP）在缺乏内源性亚基8的aap 1寄主酵母菌体内的异位表达导致转化菌株T475的生物能量功能部分恢复。导入和组装研究采用来自菌株YGL-1亚基8部分缺失的靶线粒体进行；这种控制消耗先前已被证明是作为前体N9L/Y8-1从体外进口的全长亚基8的有效组装（免疫化学监测）所必需的。结果发现，体外合成的N9L/Y8-1（K47-STP）被成功导入YGL-1线粒体，但在这些线粒体或其他被测线粒体中未观察到截断的亚基8的显著组装。T475线粒体的生物能缺陷归因于亚单位-8变体在体内的组装受损，这是由Lys47的截断造成的。因此，T475线粒体表现为亚基8的部分缺失。分离的T475线粒体能够为全长N9L/Y8-1加工后的亚基8的有效导入和组装提供载体，这一能力支持了这一结论。作为截断的子单元8分析的一部分，已经讨论了导入和组装的两个相关方面。首先，通过异体表达N9L/Y8-1重组的aap 1 mit-突变株T2-1的线粒体也被发现对来自进口N9L/Y8-1的亚基8的体外组装有效。这表明体内N9L/Y8-1异体表达导致线粒体内8亚基缺失，这可能是与野生型酵母相比，T2-1线粒体能量偶联恢复不完全的原因。其次，将N9L/Y8-2（含有与N9L直接融合的亚基8）在aap 1 mit-宿主中异体表达，产生了一个拯救的转化菌株T10-1，其表面上表现出与T475相似的生物能缺陷。尽管N9L/Y8-2在体外的输入相对较弱，但加工亚基8清楚地组装到分离的YGL-1线粒体的ATP合酶中。这表明体外组装系统能够区分亚基8的变体，如N9L/Y8-1（K47-STP）和N9L/Y8-2的组装特性。
This paper deals with a truncated derivative of subunit 8 of yeast mitochondrial ATP synthase in which a conserved positively charged residue (Lys47) has been removed by site-directed mutagenesis together with the C-terminal residue (Leu48). This derivative has been expressed as a chimaeric precursor N9L/Y8-1(K47-STP) carrying an N-terminal cleavable leader sequence (N9L), fused by a short bridging sequence to the truncated subunit-8 passenger protein. Allotopic expression of N9L/Y8-1 (K47-STP) in vivo in an aap 1 mit- host yeast strain lacking endogenous subunit 8 leads to partial restoration of bioenergetic function in the transformant strain denoted T475. Import and assembly studies were carried out in vitro using target mitochondria from strain YGL-1 partially depleted in subunit 8; such controlled depletion has been previously shown to be required for the efficient assembly (monitored immunochemically) of full-length subunit 8 imported in vitro as the precursor N9L/Y8-1. It was found that N9L/Y8-1(K47-STP) synthesized in vitro was imported successfully into YGL-1 mitochondria, but no significant assembly of the truncated subunit 8 was observed in these or any other mitochondria tested. The bioenergetic defects in T475 mitochondria are ascribed to the impaired assembly of the subunit-8 variant in vivo, resulting from the truncation at Lys47. In consequence, T475 mitochondria behave as though partially depleted of subunit 8. This conclusion was supported by the ability of isolated T475 mitochondria to provide a vehicle for the efficient import and assembly of subunit 8 processed form full-length N9L/Y8-1. Two related aspects of import and assembly have been addressed as part of the analysis of truncated subunit 8. First, mitochondria from strain T2-1, an aap 1 mit- mutant genetically reconstituted by allotopic expression of N9L/Y8-1, were also found to be effective in the in vitro assembly of subunit 8 derived from imported N9L/Y8-1. This suggests an intramitochondrial shortage of subunit 8 delivered by allotopic expression of N9L/Y8-1 in vivo, which may underlie the incomplete restoration of energy coupling in T2-1 mitochondria compared to those of wild-type yeast. Second, on allotopic expression of N9L/Y8-2 (containing subunit 8 directly fused to N9L) in the aap 1 mit- host, a rescued transformant strain T10-1 was generated which displays bioenergetic defects superficially similar to those of T475. Processed subunit 8 clearly assembled into the ATP synthase of isolated YGL-1 mitochondria, in spite of the relatively weak import of N9L/Y8-2 in vitro. This demonstrates the ability of the in vitro assembly system to distinguish assembly properties of variants of subunit 8, such as N9L/Y8-1(K47-STP) and N9L/Y8-2.