A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.
A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins.
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DOI：
10.1083/jcb.123.1.109
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发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
Pfanner N
中科院分区：
文献类型：
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作者：
Gambill BD;Voos W;Kang PJ;Miao B;Langer T;Craig EA;Pfanner N
利用两种温度敏感型酵母突变体研究了线粒体70-kD热休克蛋白（mt-hsp70）在线粒体内外膜蛋白易位中的作用。多肽易位到突变线粒体基质的程度是用基质靶向前蛋白分析的，该前蛋白被加工肽酶切割两次。前蛋白的短氨基末端片段（40-60个氨基酸）被膜电位驱动进入基质，独立于hsp70功能，允许前序列的单次切割。在突变体mt-hsp70具有可检测的结合活性的情况下，人工展开前蛋白允许完全易位到基质中。然而，在无法检测到与mt-hsp70结合的突变体线粒体中，前蛋白的成熟部分仅转移到膜间空间。我们提出mt-hsp70在前蛋白的膜易位中发挥双重作用。(a) Mt-hsp70促进多肽链在线粒体膜上的易位展开。(b) mt-hsp70与多肽链的结合对于驱动完成基质靶向前蛋白穿过内膜的运输是必不可少的。这第二个作用与前蛋白的折叠状态无关，因此确定mt- hsp70是内膜转运机制的真正组成部分。此外，我们确定了突变的位点，并表明mt- hsp70需要一个功能性的ATP酶结构域和ATP才能与多肽链结合并驱动其转位到基质中。
The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix- targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt- hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt- hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.