The J-related segment of tim44 is essential for cell viability: a mutant Tim44 remains in the mitochondrial import site, but inefficiently recruits mtHsp70 and impairs protein translocation.
Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Delta18 in addition to the endogenous wild-type Tim44. Tim44Delta18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Delta18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Delta18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.
Merlin
A
A
Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany.
Voos
W
W
Maarse
A C
AC
Meijer
M
M
Pfanner
N
N
Rassow
J
J
D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D008928:Mitochondria / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000502:physiology* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; C077016:TIM44 protein, S cerevisiae