SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk

Peschke, Markus; Goff, Mélanie Le; Koningstein, Gregory M.; Karyolaimos, Alexandros; Gier, Jan‐Willem de; Ulsen, Peter van; Luirink, Joen

doi:10.1016/j.jmb.2017.12.004

Cited by 29 publications

(24 citation statements)

References 42 publications

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“…This is deduced from the observation that SRP maintains contact to YohP even if the ribosome is dissociated by puromycin (Fig 6 and S6 Fig) and by SRP-dependent insertion of YohP after translation has been terminated by chloramphenicol (Fig 4). A putative posttranslational role of SRP has been suggested for some eukaryotic and bacterial tail-anchored (TA) proteins [69][70][71][72][73]. However, at least some bacterial TA proteins, like SciP [73], contain N-terminal amphipathic helices that could serve as cotranslational recognition sites for SRP [74,75].…”

Section: Discussionmentioning

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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Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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“…To test for authentic membrane insertion including full translocation of the short C-terminal tail, we added a short 13-residue extension in the form of an opsin tag to the C termini of NG-WALP-B/C/G. This tag is derived from the N-terminal domain of bovine opsin and known to be compatible with translocation across the bacterial inner membrane (12). These derivatives were used to investigate the translocation of the C-terminal tail across the inner membrane using a protease protection assay.…”

Section: Resultsmentioning

confidence: 99%

“…We have recently identified DjlC and Flk as bona fide E. coli TAMPs, the TMDs of which are required and sufficient for membrane targeting and insertion (12). The SRP, its membrane receptor FtsY, the membrane insertase YidC, and the cytoplasmic chaperone DnaK appeared required for optimal targeting and membrane insertion of these proteins (12).…”

Section: Introductionmentioning

confidence: 99%

“…coli TAMPs, the TMDs of which are required and sufficient for membrane targeting and insertion (12). The SRP, its membrane receptor FtsY, the membrane insertase YidC, and the cytoplasmic chaperone DnaK appeared required for optimal targeting and membrane insertion of these proteins (12). Additionally, results indicated that the comparatively more hydrophobic TMD of Flk imposed a stronger dependency on SRP than that seen with the less hydrophobic TMD of DjlC.…”

Section: Introductionmentioning

confidence: 99%

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Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

Peschke

Goff

Koningstein

et al. 2019

mBio

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Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented. IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus “regular” membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

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“…Furthermore, it has been shown that other chaperones are responsible for TA protein targeting in bacteria, at least in E. coli. 44 Considering that ScGet3 can act as a more general chaperone under specific conditions, 38 that a large group of bacterial Get3 homologs have an α-crystallin domain with expected chaperone activity, and that all the TRC40-insert-containing homologs mentioned above are predicted to have a hydrophobic groove, it is possible that these homologs act more as general chaperones than TA protein targeting factors. From this perspective, the TA protein targeting activity of cytoplasmic eukaryotic Get3 homologs could represent an adaptation of an ancient, more general chaperoning function.…”

Section: Previously Unnoticed Bacterial Get3 Homologsmentioning

confidence: 99%

The natural history of Get3‐like chaperones

Farkas

Laurentiis

Schwappach

2019

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Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail‐anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40‐insert and the hydrophobic groove essential for tail‐anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40‐insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40‐insert is present in all domains of life, we suggest that its presence does not automatically predict a tail‐anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40‐insert but have not been demonstrated to function in tail‐anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.

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SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk

Cited by 29 publications

References 42 publications

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

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

Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli

The natural history of Get3‐like chaperones

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