Gambill B D, Voos W, Kang P J, Miao B, Langer T, Craig E A, Pfanner N
Department of Biomolecular Chemistry, University of Wisconsin-Madison 53706.
J Cell Biol. 1993 Oct;123(1):109-17. doi: 10.1083/jcb.123.1.109.
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.
利用两个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）在蛋白质跨线粒体外膜和内膜转运中的作用。使用一种被加工肽酶切割两次的靶向基质的前体蛋白来分析多肽转运到突变线粒体基质中的程度。前体蛋白的短氨基末端片段（40-60个氨基酸）由膜电位驱动进入基质，这与hsp70功能无关，使得前序列仅发生一次切割。在前体蛋白人工展开的情况下，如果突变的mt-hsp70具有可检测的结合活性，则允许其完全转运到基质中。然而，在无法检测到与mt-hsp70结合的突变线粒体中，前体蛋白的成熟部分仅转运到膜间隙。我们提出mt-hsp70在转运前体蛋白的膜转运中发挥双重作用。（a）Mt-hsp70促进多肽链展开以便跨线粒体膜转运。（b）mt-hsp70与多肽链的结合对于驱动靶向基质的前体蛋白穿过内膜完成转运至关重要。这第二个作用与前体蛋白的折叠状态无关，因此将mt-hsp70鉴定为内膜转运机制的一个真正组成部分。此外，我们确定了突变位点，并表明mt-hsp70与多肽链结合并驱动其转运到基质中既需要功能性ATP酶结构域也需要ATP。
