SecYEG: Plug-and-Play!

Bacteriën bezitten één of meerdere membraanlaagjes die het binnenste van de cel scheiden van de buitenwereld. Vaak moeten eiwitten, die in de cel geproduceerd worden, door het binnenste membraan van de cel getransporteerd worden om hun functie buiten de cel te kunnen uit te oefenen. Dit proces vindt paats middels een gespecialiseerd transportmechanisme, met als centrale component het SecYEG eiwit dat een watergevuld kanaal door de membraan vormt. Het motor eiwit SecA bindt aan SecYEG en op een actief manier geleidt het te transporteren eiwit actief door het kanaal. In dit proefschrift ligt de nadruk hoe het SecYEG kanaal zich opent om het membraan transport van eiwitten mogelijk te maken. SecYEG heeft een zandlopervorm, waarbij de uitgang wordt geblokkeerd door een plug dat een eiwitsegment is van SecY dat terugvouwt in het kanaal. In dit onderzoek is onder andere vastgesteld in hoeverre de plug zich moet verplaatsen om eiwittransport mogelijk te maken. Door de plug vast te zetten in het kanaal en vervolgens eiwittransport te meten, zijn we tot de conclusie gekomen dat de plug zich maar minimaal zijwaarts verplaatst om plaats te maken voor het te transporteren eiwit. De verplaatsing van de plug hebben we ook onderzocht middels fluorescentiemethoden waarmee vastgesteld kan worden of de pluf zich een waterige omgeving bevind of gewoon onderdeel uit maakt van het kanaal. Bij eiwittransport vonden wij we dat de plug onder translocatiecondities blootgesteld wordt aan water. Echter als we kijken naar eiwitinsertie, waar het eiwit niet door het membraan gaat maar zijwaarts vanuit het kanaal in het membraan in glijdt, zagen wij geen beweging van de plug. Dit wijst erop dat de plug meehelpt om eiwitten op de goede plek terecht te laten komen, in dit geval in de membraan of buiten de membraan. In bacteria, the outside world is separated from the interior by only one or a few membrane layers consisting of lipids. Proteins produced in the cell often need to be transported across or into these membranes. This process is facilitated by a specialized transport mechanism, where the membrane complex SecYEG plays a central role as a protein conducting channel. The motor protein SecA actively guides the secretory protein through this channel. In this thesis, we have in particularly studied the opening mechanism of the SecYEG translocation channel. The channel has an hourglass shape, whereby the exit is blocked by a helical segment of SecY termed the plug. Among others, we assessed to what extent the plug has to move to vacate the central channel for proteins to pass. By fixing the plug at different locations and subsequently measuring protein translocation, it was concluded that the plug only exhibits a minimal movement to make way for the translocating protein. To analyze the plug dynamics we used a fluorescence based approach which could be used to probe the environment of the plug. During protein translocation, the plug was solvated by water as expected from the predicted channel opening mechanism. Surprisingly, during protein insertion, where the protein laterally inserta in the membrane layer instead of being translocated through the channel, there was no change is solvation and thus no movement of the plug. This indicates that the plug recognizes the incoming protein and will move away if the protein has to pass, but stays at its central position when a membrane protein enters the lipid bilayer.

The bacterial Sec-translocase: structure and mechanism

Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.

Immobilization of the Plug Domain Inside the SecY Channel Allows Unrestricted Protein Translocation

The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation.

Energy coupling efficiency in the Type I ABC transporter GlnPQ

Solute transport via ABC importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule FRET measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently-linked SBDs, which may aid our understanding of Type I ABC importers in general

Characterization of the supporting role of SecE in protein translocation

<p>SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic a-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.</p>