title: Architecture of the TIM23 inner mitochondrial translocon and interactions with the matrix import motor.
abstract: Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by action of the matrix-localized multi-subunit import motor, which is associated with the TIM23 translocon. The architecture of the import apparatus is not well understood. Here, we report results of site-specific in vivo photocross-linking along with genetic and coimmunoprecipitation analyses dissecting interactions between import motor subunits and the translocon. The translocon is composed of the two integral membrane proteins Tim23 and Tim17, each containing four membrane-spanning segments. We found that Tim23 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane domains 1 and 2 (loop 1) cross-linked to Tim44. Alterations in this loop destabilized interaction of Tim44 with the translocon. Analogously, Tim17 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane segments 1 and 2 (loop 1) cross-linked to Pam17. Alterations in this loop caused destabilization of the interaction of Pam17 with the translocon. Substitution of individual photoactivatable residues in Tim44 and Pam17 in regions we previously identified as important for translocon association resulted in cross-linking to Tim23 and Tim17, respectively. Our results are consistent with a model in which motor association is achieved via interaction of Tim23 with Tim44, which serves as a scaffold for association of other motor components, and of Tim17 with Pam17. As both Tim44 and Pam17 have been implicated as regulatory subunits of the motor, this positioning is conducive for responding to conformational changes in the translocon upon a translocating polypeptide entering the channel.
authors.affiliation: From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: See-Yeun
authors.initials: SY
authors.lastname: Ting
authors.affiliation: From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schilke
authors.affiliation: From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Masaya
authors.initials: M
authors.lastname: Hayashi
authors.affiliation: From the Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706 ecraig@wisc.edu.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Craig
mesh_terms: D000595:Amino Acid Sequence; D003600:Cytosol / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; 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 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D055668:Photochemical Processes; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D020409:Molecular Motor Proteins; C503873:Pam17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C091191:TIM17 protein, S cerevisiae; C083081:TIM23 protein, S cerevisiae; C077016:TIM44 protein, S cerevisiae
keywords: Mitochondria; Protein Cross-linking; Protein Translocation; Protein-Protein Interaction; TIM23 Complex; Yeast