Protein Conducting Nanopores

Protein Conducting Nanopores Richard Wagner Biophysics, Department of Biology/Chemistry, University of Osnabrück, Germany It is generally accepted that proteins with their numerous hydrophilic and charged amino acid side chains move across the hydrophobic phospholipid bilayer as mainly unfolded polypeptide chains through hydrophilic, hetero-oligomeric transmembrane channels composed of integral membrane proteins. First evidences for this came from the mammalian ER which was by electrophysiological techniques shown to contain ion channels with a conductance of ~220 pS (50 mM KCl) that can be activated by puromycin/KCl treatment of rough microsomal vesicles (Simon & Blobel, 1991). The first report on the reconstitution of a heterologously expressed pore forming subunit from a protein translocation complex was on Toc75 from the chloroplastic outer membrane. The channel properties of which were determined with the planar bilayer technique (Hinnah et al, 1997). The second unequivocally identified protein translocation subunit from our lab was Tom40 from the outer mitochondrial membrane (Hill et al, 1998)). Subsequently, we identified Tim23 (Truscott et al, 2001)) as well as Tim22 (Kovermann et al, 2002) as the protein translocation (Tim23) and insertion channel (Tim22) at the inner mitochondrial membrane and characterized their single channel properties using the planar bilayer technique. Using the same approaches with recombinant proteins and biochemically purified proteins from their original membranes, we could demonstrate that Tic110 forms the protein translocation channel at the inner chloroplast membrane (Heins et al, 2002). Finally we could also prove through reconstitution of the biochemically purified protein translocation complex that the puromycin/KCl activated channel in rough microsomal-vesicles (RM–vesicles) is formed by the heterotrimeric Sec61 complex (Wirth et al, 2003). The channels of the different protein translocation complexes mentioned so far displayed pore sizes which are only compatible with the transport of mainly unfolded polypeptides. In contrast to this, in peroxisomes we observed the formation of a large channel pore of Pex5p together with its docking partner Pex14p, which can be opened up to a diameter of ~9 nm, thus allowing the passage of folded proteins (Meinecke et al, 2010) . In my talk I will particularly focus on -and highlight the common biophysical properties and functions of the protein conducting nanopores from the different organelles. In addition recent results on peptide transport through the Tom40 channel will be presented in some detail. References Heins L, Mehrle A, Hemmler R, Wagner R, Kuchler M, Hormann F, Sveshnikov D, Soll J (2002) The preprotein conducting channel at the inner envelope membrane of plastids. EMBO Journal 21: 2616-2625 Hill K, Model K, Ryan MT, Dietmeier K, Martin F, Wagner R, Pfanner N (1998) Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment]. Nature 395: 516-521 Hinnah SC, Hill K, Wagner R, Schlicher T, Soll J (1997) Reconstitution of a chloroplast protein import channel. The EMBO journal 16: 7351-7360 Kovermann P, Truscott KN, Guiard B, Rehling P, Sepuri NB, Muller H, Jensen RE, Wagner R, Pfanner N (2002) Tim22, the essential core of the mitochondrial protein insertion complex, forms a voltage-activated and signal-gated channel. Mol Cell 9: 363-373 Meinecke M, Cizmowski C, Schliebs W, Kruger V, Beck S, Wagner R, Erdmann R (2010) The peroxisomal importomer constitutes a large and highly dynamic pore. Nat Cell Biol 12: 273-277 Simon SM, Blobel G (1991) A Protein-Conducting Channel in the Endoplasmic Reticulum. Cell 65: 371-380 Truscott KN, Kovermann P, Geissler A, Merlin A, Meijer M, Driessen AJ, Rassow J, Pfanner N, Wagner R (2001) A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23. Nat Struct Biol 8: 1074-1082 Wirth A, Jung M, Bies C, Frien M, Tyedmers J, Zimmermann R, Wagner R (2003) The Sec61p complex is a dynamic precursor activated channel. Mol Cell 12: 261-268