Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.
The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.
Baker
Michael J
MJ
Department of Biochemistry, La Trobe University, Melbourne 3086, Victoria, Australia.
Webb
Chaille T
CT
Stroud
David A
DA
Palmer
Catherine S
CS
Frazier
Ann E
AE
Guiard
Bernard
B
Chacinska
Agnieszka
A
Gulbis
Jacqueline M
JM
Ryan
Michael T
MT
D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility / Q000187:drug effects; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; C107250:TIM10 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae; D013762:Tetrahydrofolate Dehydrogenase