Assembly of the mitochondrial Tim9-Tim10 complex: a multi-step reaction with novel intermediates.
Protein assembly is a crucial process in biology, because most proteins must assemble into complexes to perform their function in the cell. The mitochondrial Tim9-Tim10 translocase complex, located in the mitochondrial intermembrane space, plays an essential chaperone-like role during the import of mitochondrial membrane proteins. The complex consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While structural and functional studies have indicated a dynamic nature of the complex, little is known about the assembly process and the mechanism of its function. Here we investigated the assembly process of yeast Tim9-Tim10 complex in real time, using stopped-flow fluorescence coupled with Trp mutagenesis, and stopped-flow light scattering techniques. We show that different parts of the proteins are assembled at different rates; also assembly intermediates consisting four subunits arise transiently before formation of the final hexameric Tim9-Tim10 complex. Interestingly, the assembly intermediate has more organised N-terminal helices that form an inner layer of the complex, but not the C-terminal helices, which form the outer layer of the complex. In addition, using analytical ultracentrifugation techniques, we show that Tim9 forms a homo-dimer while Tim10 is a monomer. A four-step assembly pathway of Tim9-Tim10 complex, involving formation of hetero-dimer and tetramer assembly intermediates, is proposed. This study provides the first description of the assembly pathway of this translocase complex, and insight into the mechanism of its function.
Ivanova
Ekaterina
E
Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
Jowitt
Thomas A
TA
Lu
Hui
H
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000235:genetics; D014461:Ultracentrifugation
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; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C107250:TIM10 protein, S cerevisiae; C116366:Tim9 protein, S cerevisiae; D014364:Tryptophan; D005982:Glutathione Transferase; D003545:Cysteine