Abstract. 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 aminoterminal 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.

No abstract

Abstract. To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome be (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the aminoterminal 167 residues of the precursor of cyt. be and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. be portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. be portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function.

The 70-kDa heat shock proteins (hsp7Os) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate protens coupled to cycles ofATP hydrolysis. In the prokaryote Esckerichia coi, the hsp7O DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp7Os and DnaJ-related proteins have been identified in' eukaryotes, to our knowledge no GrpErelated proteins have been reported. We report the isolation and characterization ofa eukaryotic grpE-related gene, MfGE.

Different surgical approaches for the treatment of localized prostate cancer affect the shortterm QoL results in different ways. Urinary, sexual, and bowel function and bother are affected to a similar degree by ORP, LRP, and dVP. In an older population, the tissue destruction resulting from PCryo appears to relieve obstructive and irritative urinary symptoms but at the sacrifice of sexual function compared with (103)Pd.