Preprotein Translocase of the Outer Mitochondrial Membrane: Reconstituted Tom40 Forms a Characteristic TOM Pore
Biophysik, Universität Osnabrück, FB Biologie/Chemie, D-49034 Osnabrück, Germany. Journal of Molecular Biology
(Impact Factor: 4.33).
12/2005; 353(5):1011-20. DOI: 10.1016/j.jmb.2005.09.019
Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.
Available from: Nadine Flinner
- "The analysis of purified Tom40 protein and of the TOM core complex after reconstitution into planar lipid bilayers indicated that Tom40 forms a cation selective membrane pore with an estimated inner pore diameter of 20 to 25 Å     . At high voltages, the channel revealed a complex gating behavior indicating fluctuation between different conformational states   . As expected, mitochondrial pre-sequence peptides were significantly more potent in blocking the pore than the synthetic non-mitochondrial model peptides . "
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ABSTRACT: Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with β-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of approximately 270 different Tom40 and approximately 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.
Available from: Anke Harsman
- "The addition of such peptides (in nano-to micromolar concentrations) to the electrolyte solutions in the measurement chamber can result in short blocks of the ionic current, arising from peptide binding to the pore or the passage of charged peptides, which are electrophoretically driven through the pore by the applied holding potential (Fig. 3C). This has been observed for many of the protein translocases studied in our lab (Becker et al., 2005; Hill et al., 1998; Hinnah et al., 1997; Kovermann et al., 2002; Kutik et al., 2008; Meinecke et al., 2006; Rehling et al., 2003; Truscott et al., 2001; van Wilpe et al., 1999; Wirth et al., 2003). Performing these experiments , one has to pay attention to the impact of salt concentration and pH on the properties of the peptide and pore in question. "
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ABSTRACT: Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.
Available from: Thomas Becker
- "β-barrel proteins are characteristic for the outer membranes of Gram-negative bacteria, mitochondria , and chloroplasts (Wimley, 2003; Gentle et al., 2005; Ruiz et al., 2006; Knowles et al., 2009; Walther and Rapaport, 2009; Schleiff and Becker, 2011). Mitochondrial β-barrel proteins are essential for cell viability, since the central channel-forming component of the translocase of the outer membrane (TOM) is a β-barrel protein termed Tom40 (Hill et al., 1998; Suzuki et al., 2004; Becker et al., 2005). The TOM complex functions as the general entry gate for most mitochondrial proteins synthesized in the cytosol (Ryan et al., 2000; Mihara, 2003; Johnson and Jensen, 2004; Koehler, 2004; Dolezal et al., 2006; Neupert and Hermann, 2007; Chacinska et al., 2009; Endo and Yamano, 2009; Schmidt et al., 2010). "
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ABSTRACT: The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of β-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of β-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of β-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds β-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of β-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.
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