Organization and Function of the Small Tim Complexes Acting along the Import Pathway of Metabolite Carriers into Mammalian Mitochondria*

Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria. In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins. Here, we show that the human small proteins form two distinct hetero-oligomeric complexes. A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa. This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12. Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space. Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes. During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner. For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase. However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22. We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes.

[1]  D. Mokranjac,et al.  Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria , 2003, The EMBO journal.

[2]  V. Trézéguet,et al.  The Dynamic Dimerization of the Yeast ADP/ATP Carrier in the Inner Mitochondrial Membrane Is Affected by Conserved Cysteine Residues* , 2003, Journal of Biological Chemistry.

[3]  E. Craig,et al.  Regulated Cycling of Mitochondrial Hsp70 at the Protein Import Channel , 2003, Science.

[4]  Albert Sickmann,et al.  Protein Insertion into the Mitochondrial Inner Membrane by a Twin-Pore Translocase , 2003, Science.

[5]  H. Prokisch,et al.  Tim50, a novel component of the TIM23 preprotein translocase of mitochondria , 2003, The EMBO journal.

[6]  N. Pfanner,et al.  The Mitochondrial Presequence Translocase An Essential Role of Tim50 in Directing Preproteins to the Import Channel , 2002, Cell.

[7]  S. Nishikawa,et al.  Tim50 Is a Subunit of the TIM23 Complex that Links Protein Translocation across the Outer and Inner Mitochondrial Membranes , 2002, Cell.

[8]  R. Jensen,et al.  Protein import into and across the mitochondrial inner membrane: role of the TIM23 and TIM22 translocons. , 2002, Biochimica et biophysica acta.

[9]  M. Ryan,et al.  Import and assembly of proteins into mitochondria of mammalian cells. , 2002, Biochimica et biophysica acta.

[10]  Koji Okamoto,et al.  The protein import motor of mitochondria: a targeted molecular ratchet driving unfolding and translocation , 2002, The EMBO journal.

[11]  K. Gerbitz,et al.  The C66W Mutation in the Deafness Dystonia Peptide 1 (DDP1) Affects the Formation of Functional DDP1·TIM13 Complexes in the Mitochondrial Intermembrane Space* , 2002, The Journal of Biological Chemistry.

[12]  W. Oppliger,et al.  The Tim9p–Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier , 2002, The EMBO journal.

[13]  L. Tranebjaerg,et al.  Human deafness dystonia syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex. , 2002, Human molecular genetics.

[14]  A. Merlin,et al.  A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23 , 2001, Nature Structural Biology.

[15]  K. Gerbitz,et al.  Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria* , 2001, The Journal of Biological Chemistry.

[16]  W. Oppliger,et al.  The Essential Function of the Small Tim Proteins in the TIM22 Import Pathway Does Not Depend on Formation of the Soluble 70-Kilodalton Complex , 2001, Molecular and Cellular Biology.

[17]  N. Pfanner,et al.  Versatility of the mitochondrial protein import machinery , 2001, Nature Reviews Molecular Cell Biology.

[18]  N. Pfanner,et al.  The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria , 2001, The EMBO journal.

[19]  M. Bauer,et al.  The role of the TIM8–13 complex in the import of Tim23 into mitochondria , 2000, EMBO Journal.

[20]  N. B. Sepuri,et al.  Two Intermembrane Space Tim Complexes Interact with Different Domains of Tim23p during Its Import into Mitochondria , 2000, The Journal of cell biology.

[21]  W. Oppliger,et al.  Tim18p, a New Subunit of the TIM22 Complex That Mediates Insertion of Imported Proteins into the Yeast Mitochondrial Inner Membrane , 2000, Molecular and Cellular Biology.

[22]  N. B. Sepuri,et al.  Tim18p is a new component of the Tim54p-Tim22p translocon in the mitochondrial inner membrane. , 2000, Molecular biology of the cell.

[23]  K. Gerbitz,et al.  The mitochondrial TIM22 preprotein translocase is highly conserved throughout the eukaryotic kingdom , 1999, FEBS letters.

[24]  D. Vetrie,et al.  The human family of Deafness/Dystonia peptide (DDP) related mitochondrial import proteins. , 1999, Genomics.

[25]  C. Koehler,et al.  How membrane proteins travel across the mitochondrial intermembrane space. , 1999, Trends in biochemical sciences.

[26]  C. Koehler,et al.  Different import pathways through the mitochondrial intermembrane space for inner membrane proteins , 1999, The EMBO journal.

[27]  W. Neupert,et al.  The TIM17·23 preprotein translocase of mitochondria: composition and function in protein transport into the matrix , 1999, The EMBO journal.

[28]  W. Neupert,et al.  Transport of the ADP/ATP carrier of mitochondria from the TOM complex to the TIM22·54 complex , 1999, The EMBO journal.

[29]  A. Reichert,et al.  Genetic and structural characterization of the human mitochondrial inner membrane translocase. , 1999, Journal of molecular biology.

[30]  S. Merchant,et al.  Human deafness dystonia syndrome is a mitochondrial disease. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[31]  W. Neupert,et al.  Tim9, a new component of the TIM22·54 translocase in mitochondria , 1999, The EMBO journal.

[32]  S. Merchant,et al.  Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins , 1998, The EMBO journal.

[33]  W. Neupert,et al.  Carrier protein import into mitochondria mediated by the intermembrane proteins Tim10/Mrs11 and Tim12/Mrs5 , 1998, Nature.

[34]  C. Koehler,et al.  Import of mitochondrial carriers mediated by essential proteins of the intermembrane space. , 1998, Science.

[35]  Maithreyan Srinivasan,et al.  The Tim54p–Tim22p Complex Mediates Insertion of Proteins into the Mitochondrial Inner Membrane , 1997, The Journal of cell biology.

[36]  M. Bauer,et al.  Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22 , 1996, Nature.

[37]  M. Bauer,et al.  Role of Tim23 as Voltage Sensor and Presequence Receptor in Protein Import into Mitochondria , 1996, Cell.

[38]  D. Vetrie,et al.  A novel X–linked gene, DDP, shows mutations in families with deafness (DFN–1), dystonia, mental deficiency and blindness , 1996, Nature Genetics.

[39]  Walter Neupert,et al.  The MIM complex mediates preprotein translocationacross the mitochondrial inner membrane and couples it to the mt-Hsp70/ATP driving system , 1995, Cell.

[40]  W. Neupert,et al.  Mitochondrial heat shock protein 70, a molecular chaperone for proteins encoded by mitochondrial DNA , 1994, The Journal of cell biology.

[41]  W. Neupert,et al.  Distinct steps in the import of ADP/ATP carrier into mitochondria. , 1987, The Journal of biological chemistry.

[42]  W. Neupert,et al.  Import of mitochondrial proteins. , 1984, Biochemical Society transactions.