Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix

• Published in: The Journal of cell biology Vol. 123; no. 1; pp. 119 - 126
• Main Authors: Voos, W, Gambill, B.D, Guiard, B, Pfanner, N, Craig, E.A
• Format: Journal Article
• Language: English
• Published: New York, NY Rockefeller University Press 01.10.1993; The Rockefeller University Press
• Subjects: Biochemistry; Biological and medical sciences; Biological Transport; Cell Compartmentation; Cell physiology; Cellular biology; Cytochromes; ESTRES TERMICO; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Heat shock proteins; Heat-Shock Proteins - metabolism; Imports; L-Lactate Dehydrogenase (Cytochrome); L-Lactate Dehydrogenase - genetics; L-Lactate Dehydrogenase - metabolism; Mathematical functions; MEMBRANAS CELULARES; Membrane and intracellular transports; MEMBRANE CELLULAIRE; METABOLISME DES PROTEINES; METABOLISMO PROTEICO; Mitochondria; MITOCHONDRIE; MITOCONDRIA; Molecular and cellular biology; P branes; Protein Denaturation; Protein precursors; Protein Precursors - genetics; Protein Precursors - metabolism; Protein Sorting Signals - genetics; Protein Sorting Signals - metabolism; Protein transport; PROTEINAS; PROTEINE; Proteins; Recombinant Fusion Proteins - metabolism; Reticulocytes; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; STRESS THERMIQUE; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - metabolism; Yeasts; Translocation; Mitochondria; Yeast; Membrane transport; Heat shock protein; Unfolding; Mutation
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.123.1.119

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 inter-membrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 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. b2 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