PRESEQUENCE AND MATURE PART OF PREPROTEINS STRONGLY INFLUENCE THE DEPENDENCE OF MITOCHONDRIAL PROTEIN IMPORT ON HEAT-SHOCK PROTEIN-70 IN THE MATRIX

To test the hypothesis that 70-kD mitochondrial heat shock protein (mt -hsp70) has a dual role in membrane translocation of preproteins we sc reened preproteins in an attempt to find examples which required eithe r only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal porti ons of the intermembrane space protein cytochrome b2 (cyt. b2) fused t o dihydrofolate reductase (DHFR) were differentially imported into mit ochondria containing mutant hsp70s. A fusion protein between the amino -terminal 167 residues of the precursor of cyt. b2 and DHFR was effici ently transported into mitochondria independently of both hsp70 functi ons. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the u nfoldase function of mt-hsp70, presumably caused by a conformational r estriction of the heme-bound preprotein. In the absence of heme the no ncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 por tion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the inter membrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was stri ctly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane trans location and indicate that preproteins with an intermembrane space sor ting signal can be correctly imported even in mutants with severely im paired hsp70 function.