Monitoring the protein translocation complex (SecYEG) in action
Sprache des Vortragstitels:
Englisch
Original Kurzfassung:
The bacterial SecY complex acts as a transmembrane pore for secretory proteins, and allows hydrophobic segments to pass through its lateral gate into the lipid phase. The motor protein SecA assists the translocation of hydrophilic proteins. We have monitored its binding to the reconstituted SecY complex and its subsequent release by high speed AFM. During some of these binding events we observed the insertion of SecA?s so called two-helix finger into the SecY-channel by lanthanide resonance energy transfer between a fluorescent label on the fingertip and Tb3+ in a genetically engineered binding pocket on SecY. Deep penetrations of the finger resulted in relocation of SecY?s
plug as indicated by measurements of the distance between fingertip and plug. The ??plug?? is a short helix that is (i) normally located in the center of the channel adjacent to its hydrophobic constriction zone (pore ring) and (ii) blocks the passage of small molecules through the idle SecY complex (1).
Observation of ion channel activity confirmed that the plug has moved out of the pore. We likewise observed channel activity upon binding of ribosomes, preproteins, or signal peptides (2) or upon stalling of a translocation intermediate. The latter observation contrasts with the common believe that both occupancy of the pore by a polypeptide chain and its enclosure by the pore ring in a ?gasket-like? seal maintain the membrane barrier to ions. Physiological values of the membrane potential were required to close the channel (3). This observation suggests that the polypeptide chain moves out of the aqueous pore in de-energized membranes. Cysteine cross-links between stalled translocation intermediates and SecY confirmed the finding. We conclude that the current transport model needs to be revised to account for SecY's voltage sensitivity.
Sprache der Kurzfassung:
Englisch
Vortragstyp:
Hauptvortrag / Eingeladener Vortrag auf einer Tagung
Forschungs-