A single Sec61-complex functions as a protein-conducting channel.
During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.
Kalies
Kai-Uwe
KU
Institute of Biology, CSCM, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. kukalies@gmx.de
Stokes
Vivica
V
Hartmann
Enno
E
D000818:Animals; D004285:Dogs; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D018390:Gene Targeting; D007473:Ion Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism*; D010441:Peptide Chain Elongation, Translational; D014176:Protein Biosynthesis; D021381:Protein Transport; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure*; D000069816:SEC Translocation Channels; D014158:Transcription, Genetic
D007473:Ion Channels; D008565:Membrane Proteins; D000069816:SEC Translocation Channels