In Vitro Analysis of the Bacterial Twin-Arginine-Dependent Protein Export

Moser, Michael J.; Panahandeh, Sascha; Holzapfel, Eva; Müller, Matthias

doi:10.1007/978-1-59745-466-7_5

Cited by 30 publications

(44 citation statements)

References 22 publications

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“…Protein translocation was analyzed by proteinase K resistance [31]. Immunoprecipitations were performed as described [13] except that after denaturation with SDS, samples were freed of precipitated material by centrifugation in a tabletop microfuge.…”

Section: Methodsmentioning

confidence: 99%

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

2014

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The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.

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Section: Methodsmentioning

confidence: 99%

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

2014

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“…Cells were grown overnight in the presence of 10 μg/ml tetracycline (SL119) and 35 μg/ml chloramphenicol (Top10, pSup-BpaRS-6TRN(D286R), respectively, and were used to inoculate fresh medium without antibiotics at a 1:100 ratio. Cells were grown and S-135 cell extracts were prepared as described (Moser et al , 2007). …”

Section: Methodsmentioning

confidence: 99%

“…Coupled transcription/translation of pSufI and pTorA-PhoA from plasmid DNAs was performed as described (Moser et al , 2007). In case of pTorA-PhoA, oxidizing conditions were provided by the addition of 5 mM oxidized glutathione (GSSG) at the start of synthesis.…”

Section: Methodsmentioning

confidence: 99%

“…To assay translocation of precursors into INVs, samples were treated with proteinase K according to ref. (Moser et al , 2007). Before UV-induced cross-linking (Panahandeh et al , 2008), reactions were stopped with 0.4 mM puromycin (final concentration) for 5 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…INV were reisolated by centrifugation at 100,000 g for 30 min at 4°C and resuspended in buffer to give the same ionic conditions as before with either no further additives or an ATP-regenerating system (Moser et al , 2007). Incubation at 37°C for 30 min was followed by PK treatment and UV irradiation.…”

Section: Methodsmentioning

confidence: 99%

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TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

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Jungkamp

et al. 2010

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Involvement of PpiD in Sec-dependent protein translocation

Fürst¹,

Zhou²,

Merfort³

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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The periplasmic space in between the inner and outer membrane of Gram-negative bacteria contains numerous chaperones that are involved in the biogenesis and rescue of extra-cytosolic proteins. In contrast to most of those periplasmic chaperones, PpiD is anchored by an N-terminal transmembrane domain within the inner membrane of Escherichia coli. There it is located in close proximity to the SecY subunit of the SecYEG translocon, which is the primary transporter for secretory and membrane proteins. By site-specific cross-linking we now found the periplasmic domain of PpiD also in close vicinity to the SecG subunit of the Sec translocon and we provide the first direct evidence for a functional cooperation between PpiD and the Sec translocon. Thus we demonstrate that PpiD stimulates in a concentration-dependent manner the translocation of two different secretory proteins into proteoliposomes that had been reconstituted with sub-saturating amounts of SecYEG. In addition we found ribosome-associated nascent chains of a secretory protein stalled at SecY also being in close contact to PpiD. Collectively these results suggest that PpiD plays a role in clearing the Sec translocon of newly translocated secretory proteins thereby improving the overall translocation efficiency. Consistent with this conclusion we demonstrate that PpiD contributes to the efficient detachment of newly secreted OmpA from the inner membrane and in doing so, seems to cooperate in a hierarchical manner with other periplasmic chaperones such as SurA, DegP, and Skp.

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In Vitro Analysis of the Bacterial Twin-Arginine-Dependent Protein Export

Cited by 30 publications

References 22 publications

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

TatB Functions as an Oligomeric Binding Site for Folded Tat Precursor Proteins

Involvement of PpiD in Sec-dependent protein translocation

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