Noncompetitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in Escherichia coli

Jauß, Benjamin; Petriman, Narcis-Adrian; Drepper, Friedel; Franz, Lisa; Sachelaru, Ilie; Welte, Thomas; Steinberg, Ruth; Warscheid, Bettina; Koch, Hans‐Georg

doi:10.1074/jbc.ra119.010686

Cited by 38 publications

(69 citation statements)

References 96 publications

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“…This value seems implausibly low, so it is very likely that other factors will increase this value substantially in vivo. These might include auxiliary drivers of transport, particularly the PMF (24,25), and some of the many other proteins that associate with the Sec system, such as SecDF, PpiD, and YfgM (41,42). It does, however, make sense that all these factors affect p res but not ATP turnover itself; it is of course far easier to add additional driving forces to a ratchet than to a directly coupled motor.…”

Section: Discussionmentioning

confidence: 99%

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

Allen

Watkins

Dillingham

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity—a prerequisite for understanding how biological systems work—has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

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Section: Discussionmentioning

confidence: 99%

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

Allen

Watkins

Dillingham

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…All small membrane proteins were cloned into the arabinose-inducible pBad24 vector [ 92 ] with a His 6 -tag for in vivo expression experiments using the Gibson Assembly protocol [ 93 ] and the Gibson Assembly Cloning Kit (New England BioLabs) using 100 ng vector and a 1:3 molar vector/insert ratio. For in vitro and in vivo T7-dependent expression, yohP with and without the His 6 -coding sequence and ykgR were also cloned into the pET19b (Novagen) and pRS1 [ 94 ] expression vectors. For facilitating detection of YkgR, two methionine residues were attached to the C-terminus.…”

Section: Methodsmentioning

confidence: 99%

“…Antibodies against YfgM were a gift of Dan. O. Daley, University Stockholm, and have been validated before [41,94]. Monoclonal peroxidase-conjugated antibodies against the His6-tag (HisProbe-HRP Conjugate) were purchased from Thermo Scientific and from Roche.…”

Section: Immune Detection and Antibodiesmentioning

confidence: 99%

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

et al. 2020

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Small membrane proteins represent a largely unexplored yet abundant class of proteins in pro-and eukaryotes. They essentially consist of a single transmembrane domain and are associated with stress response mechanisms in bacteria. How these proteins are inserted into the bacterial membrane is unknown. Our study revealed that in Escherichia coli, the 27amino-acid-long model protein YohP is recognized by the signal recognition particle (SRP), as indicated by in vivo and in vitro site-directed cross-linking. Cross-links to SRP were also observed for a second small membrane protein, the 33-amino-acid-long YkgR. However, in contrast to the canonical cotranslational recognition by SRP, SRP was found to bind to YohP posttranslationally. In vitro protein transport assays in the presence of a SecY inhibitor and proteoliposome studies demonstrated that SRP and its receptor FtsY are essential for the posttranslational membrane insertion of YohP by either the SecYEG translocon or by the YidC insertase. Furthermore, our data showed that the yohP mRNA localized preferentially and translation-independently to the bacterial membrane in vivo. In summary, our data revealed that YohP engages an unique SRP-dependent posttranslational insertion pathway that is likely preceded by an mRNA targeting step. This further highlights the enormous plasticity of bacterial protein transport machineries.

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“…This value seems implausibly low, so it is very likely that other factors will increase this value substantially in vivo. These might include auxiliary drivers of transport, particularly the PMF (Brundage et al, 1990;Schiebel et al, 1991), and some of the many other proteins that associate with the Sec system such as SecDF, PpiD and YfgM (Jauss et al, 2019;Pogliano and Beckwith, 1994). It does, however, makes sense that all these factors affect p res , not ATP turnover itself:…”

Section: Discussionmentioning

confidence: 99%

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

Allen

Watkins

Dillingham

et al. 2020

Preprint

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The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity -a prerequisite for understanding how biological systems works -has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, ~30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event.Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient -in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

show abstract

Noncompetitive binding of PpiD and YidC to the SecYEG translocon expands the global view on the SecYEG interactome in Escherichia coli

Cited by 38 publications

References 96 publications

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

Posttranslational insertion of small membrane proteins by the bacterial signal recognition particle

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

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