Membrane-embedded C-terminal Segment of Rat Mitochondrial TOM40 Constitutes Protein-conducting Pore with Enriched β-Structure*

TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% β-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(ΔN165)) as a recombinant protein with 62% β-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched β-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

[1]  K. Mihara,et al.  Characterization of adrenodoxin precursor expressed in Escherichia coli. , 1992, Journal of biochemistry.

[2]  N. Pfanner,et al.  Preprotein Translocase of the Outer Mitochondrial Membrane: Molecular Dissection and Assembly of the General Import Pore Complex , 1998, Molecular and Cellular Biology.

[3]  E. Mekada,et al.  Characterization of Rat TOM40, a Central Component of the Preprotein Translocase of the Mitochondrial Outer Membrane* , 2000, The Journal of Biological Chemistry.

[4]  N. Pfanner,et al.  Protein Import Channel of the Outer Mitochondrial Membrane: a Highly Stable Tom40-Tom22 Core Structure Differentially Interacts with Preproteins, Small Tom Proteins, and Import Receptors , 2001, Molecular and Cellular Biology.

[5]  K. Mihara,et al.  In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Mihara,et al.  Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the ‘acid chain’ hypothesis , 1998, The EMBO journal.

[7]  R. Benz,et al.  The Preprotein Translocation Channel of the Outer Membrane of Mitochondria , 1998, Cell.

[8]  W. Neupert,et al.  Recognition of preproteins by the isolated TOM complex of mitochondria , 2000, The EMBO journal.

[9]  J. Reed,et al.  A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism. , 1997, Analytical biochemistry.

[10]  C E Lawrence,et al.  Detection of likely transmembrane β-strand regions in sequences of mitochondrial pore proteins using the Gibbs sampler , 1996, Journal of bioenergetics and biomembranes.

[11]  Y. Kagawa,et al.  Mitochondrial outer membrane contains a protein producing nonspecific diffusion channels. , 1980, The Journal of biological chemistry.

[12]  P. Schultz,et al.  Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  W. Neupert,et al.  The protein import apparatus of the mitochondrial outer membrane , 1995 .

[14]  Daisuke Kohda,et al.  Functions of outer membrane receptors in mitochondrial protein import. , 2002, Biochimica et biophysica acta.

[15]  T. Lithgow,et al.  Tom40, the import channel of the mitochondrial outer membrane, plays an active role in sorting imported proteins , 2003, The EMBO journal.

[16]  Holger Prokisch,et al.  Reconstituted TOM core complex and Tim9/Tim10 complex of mitochondria are sufficient for translocation of the ADP/ATP carrier across membranes. , 2003, Molecular biology of the cell.

[17]  R. Hegerl,et al.  The Tom Core Complex , 1999, The Journal of cell biology.

[18]  W. Neupert,et al.  Mitochondrial Protein Import , 1997, The Journal of Biological Chemistry.

[19]  M. Radermacher,et al.  Protein translocase of the outer mitochondrial membrane: role of import receptors in the structural organization of the TOM complex. , 2002, Journal of molecular biology.

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

[21]  F. Nargang,et al.  Characterization of Neurospora crassa Tom40-deficient Mutants and Effect of Specific Mutations on Tom40 Assembly* , 2003, The Journal of Biological Chemistry.

[22]  W. Neupert,et al.  Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria. , 2001, Molecular biology of the cell.

[23]  W. Neupert,et al.  Biogenesis of Tom40, Core Component of the Tom Complex of Mitochondria , 1999, The Journal of cell biology.

[24]  B. Dobberstein,et al.  Common Principles of Protein Translocation Across Membranes , 1996, Science.

[25]  N. Akaike,et al.  Alpha 2 adrenoceptor potentiates glycine receptor-mediated taurine response through protein kinase A in rat substantia nigra neurons. , 1996, Journal of neurophysiology.

[26]  P. Schultz,et al.  Tom40 protein import channel binds to non-native proteins and prevents their aggregation , 2003, Nature Structural Biology.

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

[28]  Walter Neupert.  Protein import into mitochondria. , 1997 .

[29]  B. Litman,et al.  Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure. , 1985, Biochimica et biophysica acta.

[30]  K. Iwata,et al.  Interaction between mitochondrial precursor proteins and cytosolic soluble domains of mitochondrial import receptors, Tom20 and Tom70, measured by surface plasmon resonance. , 1998, Biochemical and biophysical research communications.

[31]  W. Neupert,et al.  cis and trans Sites of the TOM Complex of Mitochondria in Unfolding and Initial Translocation of Preproteins* , 1998, The Journal of Biological Chemistry.

[32]  W. Neupert,et al.  Mitochondrial protein import: Reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding , 1995, Cell.

[33]  W. Neupert,et al.  Apocytochrome c requires the TOM complex for translocation across the mitochondrial outer membrane , 2001, The EMBO journal.

[34]  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.

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

[36]  T. Endo,et al.  Functional cooperation and separation of translocators in protein import into mitochondria, the double-membrane bounded organelles , 2003, Journal of Cell Science.