Unfolding of preproteins upon import into mitochondria.
Unfolding of preproteins and translocation across the mitochondrial membranes requires their interaction with mt-Hsp70 and Tim44 at the inner face of the inner membrane and ATP as an energy source. We measured the temperature dependence of the rates of unfolding and import into the matrix of two folded passenger domains, the tightly folded heme-binding domain (HBD) of cytochrome b2 and the loosely folded mouse dihydrofolate reductase (DHFR). Despite the stability of the HBD, its rates of thermal breathing were fast and the preprotein was imported rapidly at all temperatures. In contrast, rates of unfolding and import of DHFR were strongly temperature dependent and import was significantly slower than unfolding. In addition, import rates of DHFR were strongly dependent on the length of the presequence. We propose that the mitochondrial import motor does not exert a constant pulling force. Rather, mt-Hsp70 appears to release a translocating polypeptide chain such that the precursor can then slide back and refold on the surface of the mitochondria. Refolding competes with translocation, and passengers may undergo several rounds of unfolding and refolding prior to their import.
Gaume
B
B
Institut für Physiologische Chemie der Universität München, Goethestrasse 33, 80336 München, Germany.
Klaus
C
C
Ungermann
C
C
Guiard
B
B
Neupert
W
W
Brunner
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D042844:L-Lactate Dehydrogenase (Cytochrome); D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism*; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D011498:Protein Precursors; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase