Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria.-猫眼课题宝

DOI：

10.1091/mbc.12.5.1189

发表时间：

2001-05

期刊：

Molecular biology of the cell

影响因子：

3.3

通讯作者：

中科院分区：

生物学3区

文献类型：

--

作者：

D. Rapaport;R. D. Taylor;M. Käser;T. Langer;W. Neupert;F. Nargang

Tom40是线粒体膜外转位酶(TOM复合体)的主要亚基。为了研究Tom40的组装途径，我们利用粗毛脉孢菌Tom40蛋白的野生型和变异型，在体外和体内跟踪了该蛋白整合到TOM复合体中的情况。当输入到分离的线粒体时，缺少前20个或前40个氨基酸残基的Tom40前体蛋白被组装成野生型蛋白。相反，缺少41到60个残基的Tom40前体含有蛋白质的一个高度保守的区域，在组装的中间阶段被阻止。我们构建了影响这一区域的Tom40的突变版本，并将这些基因转化为包含Tom40零核的保护性异核体。表达突变型Tom40蛋白的同核菌株存在生长速度缺陷和分生孢子形成能力缺陷。用蓝色天然凝胶电泳法对这些菌株的TOM复合体进行分析，发现凝胶迁移率发生了变化，并有从复合体中丢失Tom40亚基的趋势。因此，体外和体内研究都表明，残基41到60含有Tom40正确组装/稳定进入TOM复合体所需的序列。最后，我们发现线粒体外膜上的TOM复合体能够在体外进行亚单位交换。提出了Tom40亚基整合到TOM复合体中的模型。

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.