The mammalian and yeast translocon complexes comprise a characteristic Sec61 channel Frank Erdmann a,1 , Martin Jung b , Patrick Maurer b , Anke Harsman a , Richard Zimmermann b , Richard Wagner a, * a Biophysik, Universität Osnabrück, FB Biologie/Chemie, 49076 Osnabrück, Germany b Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, 66421 Homburg, Germany article info Article history: Received 29 April 2010 Available online 5 May 2010 Keywords: Protein translocation Sec61 complex Endoplasmic reticulum abstract In eukaryotes, protein translocation across and insertion into the membrane of the endoplasmic reticu- lum (ER) is facilitated by a protein-conducting channel, the Sec61 complex or translocon. In our previous electrophysiological studies, we characterized the mammalian Sec61 channel from Canis familiaris. Here we extended these initial results to the Sec61 channel from the yeast Saccharomyces cerevisiae and com- pared the basic electrophysiological properties of both channel preparations with respect to the gating behaviour, distribution of channel open states, ionic conductance, approximated pore dimensions, rever- sal potential and selectivity as well as voltage-dependent open probability. We found that the Sec61 com- plexes from both species displayed conformable characteristics of the highly dynamic channel in an intrinsically open state. In contrast, the bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, displayed under the same experimental conditions significantly different properties residing in an intrinsically closed state. We therefore propose that considerable differences between the respective eukaryote and prokaryote protein-conducting channel units and their regulation exist. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The initial step in the biogenesis of approximately 30% of eukaryotic proteins is their integration into the membrane or their transport into the lumen of the endoplasmic reticulum (ER) [3]. Protein integration or transport into the ER can occur co- or post- translationally and typically requires signal peptides at the amino terminus of the precursor proteins and a protein translocase in the ER membrane. This protein translocase includes Sec61a (Sec61p in yeast), Sec61b (Sbh1p), and Sec61c (Sss1p) as core components [8,10,20]. In our previous electrophysiological analysis of the Sec61 com- plex in canine pancreatic microsomes or reconstituted into prote- oliposomes, we characterized mammalian Sec61 complexes containing either a mixture of undefined nascent precursor poly- peptides in transit (cotranslational transport), or a defined prese- cretory polypeptide (co- and posttranslational transport). The observed conductance properties were almost identical under these various conditions and support the concept of an oligomeric Sec61 complex with a channel opening at the subcomplex interface [28]. Here we extended this initial study to the Sec61 complexes from yeast microsomes, demonstrating that these are also highly dynamic channels with a multitude of conductance states. Further- more, upon refined analysis of channel properties, we observed that the channels from Canis familiaris and Saccharomyces cerevisiae displayed analogous, if not identical, characteristics with respect to ionic conductance, pore size, ionic selectivity, gating characteristics and voltage-dependent open probability. Moreover, the channel of the mammalian Sec61 complex resides in an intrinsically open state. In contrast to this, the channel of the purified bacterial Sec61-homologue, the SecYEG complex from Escherichia coli, re- sides in an intrinsically closed state. 2. Materials and methods 2.1. Materials CHAPS, deoxy Big CHAP, and digitonin were purchased from Calbiochem, Darmstadt, Germany. Purified phospholipids (phos- phatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine) were from Sigma, egg yolk phospholipids were from Avanti Polar lipids, and L-a phosphatidylcholine (egg) came from Larodan Fine Chemicals. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.168 * Corresponding author. Address: FB Biologie/Chemie, Universität Osnabrück, Barbarastr. 13, 49076 Osnabrück, Germany. Fax: +49 541 9692243. E-mail address: wagner@uos.de (R. Wagner). 1 Present address: Institut für Physiologie 1, Westfälische Wilhelms-Universität Münster, 48179 Münster, Germany. Biochemical and Biophysical Research Communications 396 (2010) 714–720 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc