Two intermembrane space TIM complexes interact with different domains of Tim23p during its import into mitochondria.
Tim23p (translocase of the inner membrane) is an essential import component located in the mitochondrial inner membrane. To determine how the Tim23 protein itself is transported into mitochondria, we used chemical cross-linking to identify proteins adjacent to Tim23p during its biogenesis. In the absence of an inner membrane potential, Tim23p is translocated across the mitochondrial outer membrane, but not inserted into the inner membrane. At this intermediate stage, we find that Tim23p forms cross-linked products with two distinct protein complexes of the intermembrane space, Tim8p-Tim13p and Tim9p-Tim10p. Tim9p and Tim10p cross-link to the COOH-terminal domain of the Tim23 protein, which carries all of the targeting signals for Tim23p. Therefore, our results suggest that the Tim9p-Tim10p complex plays a key role in Tim23p import. In contrast, Tim8p and Tim13p cross-link to the hydrophilic NH(2)-terminal segment of Tim23p, which does not carry essential import information and, thus, the role of Tim8p-Tim13p is unclear. Tim23p contains two matrix-facing, positively charged loops that are essential for its insertion into the inner membrane. The positive charges are not required for interaction with the Tim9p-Tim10p complex, but are essential for cross-linking of Tim23p to components of the inner membrane insertion machinery, including Tim54p, Tim22p, and Tim12p.
Davis
A J
AJ
Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Sepuri
N B
NB
Holder
J
J
Johnson
A E
AE
Jensen
R E
RE
D000596:Amino Acids / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D017353:Gene Deletion; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D016296:Mutagenesis / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C107250:TIM10 protein, S cerevisiae; C100674:TIM12 protein, S cerevisiae; C403093:TIM13 protein, S cerevisiae; C102549:TIM22 protein, S cerevisiae; C083081:TIM23 protein, S cerevisiae; C110036:TIM54 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae