Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel.

The protein insertion complex of the mitochondrial inner membrane is crucial for import of the numerous multitopic membrane proteins with internal targeting signals. Little is known about the molecular mechanism of this complex, including whether it forms a real channel or merely acts as scaffold for protein insertion. We report the unexpected observation that Tim22 is the only essential membrane-integrated subunit of the complex. Reconstituted Tim22 forms a hydrophilic, high-conductance channel with distinct opening states and pore diameters. The channel is voltage-activated and specifically responds to an internal targeting signal, but not to presequences. Thus, a protein insertion complex can combine three essential functions, signal recognition, channel formation, and energy transduction, in one central component.

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

[2]  W. Wickner,et al.  Biogenesis of the Gram-Negative Bacterial Envelope , 1997, Cell.

[3]  G. R. Smith,et al.  A novel method for structure-based prediction of ion channel conductance properties. , 1997, Biophysical journal.

[4]  W. Neupert,et al.  The carboxyl-terminal two-thirds of the ADP/ATP carrier polypeptide contains sufficient information to direct translocation into mitochondria. , 1987, The Journal of biological chemistry.

[5]  T A Rapoport,et al.  Protein transport across the eukaryotic endoplasmic reticulum and bacterial inner membranes. , 1996, Annual review of biochemistry.

[6]  T. Rapoport,et al.  The structure of ribosome-channel complexes engaged in protein translocation. , 2000, Molecular cell.

[7]  J. Walker,et al.  Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. , 1996, Journal of molecular biology.

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

[9]  B. Glick,et al.  Role of ATP in the intramitochondrial sorting of cytochrome c1 and the adenine nucleotide translocator. , 1992, The EMBO journal.

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

[11]  Reiner Hegerl,et al.  Tom40, the Pore-Forming Component of the Protein-Conducting Tom Channel in the Outer Membrane of Mitochondria , 2001, The Journal of cell biology.

[12]  W. Neupert,et al.  Transport of proteins into mitochondria: a potassium diffusion potential is able to drive the import of ADP/ATP carrier. , 1985, The EMBO journal.

[13]  O. Krasilnikov,et al.  A simple method for the determination of the pore radius of ion channels in planar lipid bilayer membranes. , 1992, FEMS microbiology immunology.

[14]  K. Dietmeier,et al.  Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins , 1998, Nature.

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

[16]  S. Cho,et al.  A simple method for a determination , 2003 .

[17]  R. Wagner,et al.  A rectifying ATP‐regulated solute channel in the chloroplastic outer envelope from pea , 1999, The EMBO journal.

[18]  N. Pfanner,et al.  The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70–Tim44 , 1997, The EMBO journal.

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

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

[21]  S. Bezrukov,et al.  The charge state of an ion channel controls neutral polymer entry into its pore , 1997, European Biophysics Journal.

[22]  M. Bauer,et al.  Protein translocation into mitochondria: the role of TIM complexes. , 2000, Trends in cell biology.

[23]  N. Pfanner,et al.  Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase , 1999, Nature.

[24]  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.

[25]  L. Hendershot,et al.  BiP Maintains the Permeability Barrier of the ER Membrane by Sealing the Lumenal End of the Translocon Pore before and Early in Translocation , 1998, Cell.

[26]  D. Laver,et al.  Interpretation of substates in ion channels: unipores or multipores? , 1997, Progress in biophysics and molecular biology.

[27]  R. Jensen,et al.  Tim23p contains separate and distinct signals for targeting to mitochondria and insertion into the inner membrane. , 1998, Molecular biology of the cell.

[28]  F. Hartl,et al.  Mitochondrial protein import. , 1987, Biochimica et biophysica acta.

[29]  R. Wagner,et al.  Reconstitution of a chloroplast protein import channel , 1997, The EMBO journal.

[30]  K. Kinnally,et al.  Tim23, a Protein Import Component of the Mitochondrial Inner Membrane, Is Required for Normal Activity of the Multiple Conductance Channel, MCC , 1997, The Journal of cell biology.

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

[32]  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.

[33]  G. Schulz,et al.  The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins. , 2000, Journal of molecular biology.

[34]  S. Merchant,et al.  How membrane proteins travel across the mitochondrial intermembrane space. , 1999, Trends in biochemical sciences.

[35]  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.

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

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

[38]  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.

[39]  C. Mannella,et al.  Conformational changes in the mitochondrial channel protein, VDAC, and their functional implications. , 1998, Journal of structural biology.

[40]  D. S. Allison,et al.  Artificial mitochondrial presequences. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[41]  J. Schneider-Mergener,et al.  Distribution of Binding Sequences for the Mitochondrial Import Receptors Tom20, Tom22, and Tom70 in a Presequence-carrying Preprotein and a Non-cleavable Preprotein* , 1999, The Journal of Biological Chemistry.

[42]  W. Neupert,et al.  Mitochondrial protein import: Nucleoside triphosphates are involved in conferring import-competence to precursors , 1987, Cell.

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

[44]  N. Pfanner,et al.  Biogenesis of Tim proteins of the mitochondrial carrier import pathway: differential targeting mechanisms and crossing over with the main import pathway. , 1999, Molecular biology of the cell.

[45]  B. Welch.  The structure , 1992 .

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

[47]  J Frank,et al.  Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex. , 1997, Science.

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

[49]  W. Neupert,et al.  Protein translocation into mitochondria , 1998, BioFactors.