2018
DOI: 10.1101/248310
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Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Abstract: Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon -SecYMKEG -resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside a combination of co… Show more

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Cited by 2 publications
(4 citation statements)
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“…In this system there is a hidden stage, once the substrate has engaged the open translocon and before the substrate is actively translocated, that lasts for roughly 5 s in the absence of the chaperone Sec B. This phase, which is independent of substrate length, is rate-limiting in the translocation of small substrates and is likely associated with protein unfolding, because the presence of the chaperone catalyzes unfolding, both reducing ATP consumption and accelerating the process 6 . Our unfolding kinetics at low voltages, although for a different substrate, are consistent with this timescale.…”
Section: Discussionmentioning
confidence: 99%
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“…In this system there is a hidden stage, once the substrate has engaged the open translocon and before the substrate is actively translocated, that lasts for roughly 5 s in the absence of the chaperone Sec B. This phase, which is independent of substrate length, is rate-limiting in the translocation of small substrates and is likely associated with protein unfolding, because the presence of the chaperone catalyzes unfolding, both reducing ATP consumption and accelerating the process 6 . Our unfolding kinetics at low voltages, although for a different substrate, are consistent with this timescale.…”
Section: Discussionmentioning
confidence: 99%
“…A fter protein synthesis in the cytosol many proteins must translocate across a membrane in order to reach their final compartment or the extracellular medium. Among them we find mitochondrial protein precursors 1 and many secretory preproteins translocated into the endoplasmic reticulum (ER) in eukaryotes [2][3][4][5] or the periplasm in prokaryotes [6][7][8] . Frequently, unfolded forms of proteins are subject to posttranslational translocation 9 through a 1-2 nanometer wide protein pore present in the lipid bilayer 5,10,11 .…”
mentioning
confidence: 99%
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“…Plug domain dynamics were also studied by an in vitro smFRET approach (35). To follow the opening of the channel, the plug and cytosolic loop of TMD2 were labeled with a donor and acceptor fluorophore, respectively.…”
Section: The Secyeg Transloconmentioning
confidence: 99%