Many proteins need to be transported across the membrane of the endoplasmic reticulum (ER)
or across the bacterial plasma membrane during or after their biosynthesis. A protein complex that is conserved throughout all kingdoms of life ?the Sec61p complex in eukaryotes and the SecYEG complex in bacteria and archaea ? represents a major transporting pathway. It acts as a transmembrane pore for secretory proteins, and allows hydrophobic segments of membrane proteins to pass through its lateral gates into the lipid phase. This complex cooperates either with ribosomes or motor proteins, as well as with a variety of other proteins that are required for different steps in the translocation process. We have now obtained mechanistic insight into the molecular details of the protein transport process by having designed minimalistic systems for protein translocation with purified and reconstituted SecYEC complexes at their core (1). We found that the assembly of the translocatio
n machinery with the motor protein SecA occurs in three steps: (i) SecA binds to the lipid bilayer and migrates along its surface as visualized by fluorescence correlation spectroscopy; (ii) It anchors at SecYEG?s negatively charged annular lipids as revealed by luminescence resonance energy transfer (LRET), and (iii) SecA binds to SecYEG as visualized by high speed atomic force microscopy of single complexes in suspended membranes (2). We also investigated how the translocon maintains the barrier to small molecules during the translocation of large proteins. We were able to rule out two hypotheses that (i) intrinsic anion selectivity or (ii) spontaneous engulfment of the translocating peptide by a hydrophic ring of amino acids is sufficient to prevent ion leakage (3). Instead, our electrophysiological experiments revealed the importance of SecYEG?s voltage driven conformational changes for barrier preservation (4). *No room for citations.
Sprache der Kurzfassung:
Hauptvortrag / Eingeladener Vortrag auf einer Tagung