Juxtaposition of the two distal CX3C motifs via intrachain disulfide bonding is essential for the folding of Tim10.
The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane. A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif. Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays). As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10. The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry. The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys44 with Cys61 and an outer pair between Cys40 and Cys65. These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects. Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process.
Allen
Scott
S
School of Biological Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
Lu
Hui
H
Thornton
David
D
Tokatlidis
Kostas
K
D020816:Amino Acid Motifs; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / 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; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D003902:Detergents; D004220:Disulfides; D029968:Escherichia coli Proteins; 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; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D013438:Sulfhydryl Compounds; C478387:TIM10 protein, E coli; C107250:TIM10 protein, S cerevisiae; C478388:TIM9 protein, E coli; C116366:Tim9 protein, S cerevisiae; D014357:Trypsin; D003545:Cysteine; D004229:Dithiothreitol