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 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.
Gambill
B D
BD
Department of Biomolecular Chemistry, University of Wisconsin-Madison 53706.
Voos
W
W
Kang
P J
PJ
Miao
B
B
Langer
T
T
Craig
E A
EA
Pfanner
N
N
D000595:Amino Acid Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D011489:Protein Denaturation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
D006360:Heat-Shock Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D006180:Proton-Translocating ATPases