A DUAL ROLE FOR MITOCHONDRIAL HEAT-SHOCK PROTEIN-70 IN MEMBRANE TRANSLOCATION OF PREPROTEINS

The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in prote in translocation across both the outer and inner mitochondrial membran es was studied using two temperature-sensitive yeast mutants. The degr ee of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twic e by the processing peptidase. A short amino-terminal segment of the p reprotein (40-60 amino acids) was driven into the matrix by the membra ne potential, independent of hsp70 function, allowing a single cleavag e of the presequence. Artificial unfolding of the preprotein allowed c omplete translocation into the matrix in the case where mutant mt-hsp7 0 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 p ropose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide ch ain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the comp letion of transport of a matrix-targeted preprotein across the inner m embrane. This second role is independent of the folding state of the p reprotein, thus identifying mt-hsp70 as a genuine component of the inn er membrane translocation machinery. Furthermore we determined the sit es 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.