title: Assembly of the mitochondrial Tim9-Tim10 complex: a multi-step reaction with novel intermediates.
abstract: 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.
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Ivanova
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jowitt
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
mesh_terms: 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
chemical_list: 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