Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering.
Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.
Schiller
Dirk
D
Department of Biochemistry, 433 Babcock Drive, University of Wisconsin-Madison, Madison, WI 53726, USA.
Cheng
Yu Chin
YC
Liu
Qinglian
Q
Walter
William
W
Craig
Elizabeth A
EA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004252:DNA Mutational Analysis; D026901:Membrane Transport Proteins / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; C077016:TIM44 protein, S cerevisiae; D000226:Mitochondrial ADP, ATP Translocases; D000252:Calcium-Transporting ATPases