Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells.
The Saccharomyces cerevisiae TIM10 complex (TIM10c) is an ATP-independent chaperone of the mitochondrial intermembrane space, involved in transport of polytopic membrane proteins. The complex is an alpha(3)beta(3) hexamer of Tim9 and Tim10 subunits. We have generated specific mutations in charged residues in the central core domain of each subunit delineated by the characteristic twin CX(3)C motif, and investigated the effect of these mutations on subunit folding, complex assembly and TIM10 function in vitro and in vivo. Any combination of mutations that included a specific glutamate residue, conserved in all known Tim9 and Tim10 sequences, abolished assembly of the TIM10 complex. In vivo complementation analyses using a MET3-TIM10 strain that is selectively inactivated for the expression of wild-type Tim10 showed that (i) an N-terminal deleted version of Tim10 that was previously shown to be defective in substrate binding is lethal under all conditions, but (ii) the charged residues mutant of Tim10 that is defective in assembly with Tim9 can restore growth in glucose, but not in non-fermentable carbon sources. These data suggest that formation of the hexamer is beneficial but not vital for TIM10 function, whilst the N-terminal substrate-binding region of Tim10 is essential in vivo.
Vergnolle
Mailys A S
MA
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
Alcock
Felicity H
FH
Petrakis
Nikos
N
Tokatlidis
Kostas
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D029701:Saccharomyces cerevisiae Proteins; C107250:TIM10 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae; D000255:Adenosine Triphosphate