Revealing the mechanisms of membrane protein export by virulence-associated bacterial secretion systems

Krampen, Lea; Malmsheimer, Silke; Grin, Iwan; Trunk, Thomas; Lührmann, Anja; Gier, Jan‐Willem de; Wagner, Samuel

doi:10.1038/s41467-018-05969-w

Cited by 31 publications

(55 citation statements)

References 51 publications

(81 reference statements)

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“…We also compared the average Gibbs free energy (Δ G app ) values that are associated with the insertion of TMDs from the translocon to the IM and that varied with the positions, lengths, and amphiphilicities of residues in the TMDs ( 50 ). Because the identified single-spanning proteins of the two classes belong mostly to the targeted class and the single-spanning proteins had lower Δ G app values than multispanning proteins ( 51 ), we further divided each class into single-spanning and multispanning classes to rectify this comparison. The TMD1s of the targeted class proteins had a Δ G app value similar to that of the untargeted class proteins ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Zhao

Cui

et al. 2021

mBio

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The signal recognition particle (SRP) is conserved in all living organisms, and it cotranslationally delivers proteins to the inner membrane or endoplasmic reticulum. Recently, SRP loss was found not to be lethal in either the eukaryote Saccharomyces cerevisiae or the prokaryote Streptococcus mutans. In Escherichia coli, the role of SRP in mediating inner membrane protein (IMP) targeting has long been studied. However, the essentiality of SRP remains a controversial topic, partly hindered by the lack of strains in which SRP is completely absent. Here we show that the SRP was nonessential in E. coli by suppressor screening. We identified two classes of extragenic suppressors—two translation initiation factors and a ribosomal protein—all of which are involved in translation initiation. The translation rate and inner membrane proteomic analyses were combined to define the mechanism that compensates for the lack of SRP. The primary factor that contributes to the efficiency of IMP targeting is the extension of the time window for targeting by pausing the initiation of translation, which further reduces translation initiation and elongation rates. Furthermore, we found that easily predictable features in the nascent chain determine the specificity of protein targeting. Our results show why the loss of the SRP pathway does not lead to lethality. We report a new paradigm in which the time delay in translation initiation is beneficial during protein targeting in the absence of SRP. IMPORTANCE Inner membrane proteins (IMPs) are cotranslationally inserted into the inner membrane or endoplasmic reticulum by the signal recognition particle (SRP). Generally, the deletion of SRP can result in protein targeting defects in Escherichia coli. Suppressor screening for loss of SRP reveals that pausing at the translation start site is likely to be critical in allowing IMP targeting and avoiding aggregation. In this work, we found for the first time that SRP is nonessential in E. coli. The time delay in initiation is different from the previous mechanism that only slows down the elongation rate. It not only maximizes the opportunity for untranslated ribosomes to be near the inner membrane but also extends the time window for targeting translating ribosomes by decreasing the speed of translation. We anticipate that our work will be a starting point for a more delicate regulatory mechanism of protein targeting.

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Section: Resultsmentioning

confidence: 99%

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Zhao

Cui

et al. 2021

mBio

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“… 2015 ; Krampen et al. 2018 ). Furthermore, binding of the Salmonella chaperone SicA was found to prevent degradation of both SipB and SipC by cytosolic proteases (Tucker and Galan 2000 ).…”

Section: Substrate Targeting and Secretionmentioning

confidence: 99%

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

Wagner

Grin

Malmsheimer

et al. 2018

FEMS Microbiology Letters

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151

134

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Virulence-associated type III secretion systems (T3SS) serve the injection of bacterial effector proteins into eukaryotic host cells. They are able to secrete a great diversity of substrate proteins in order to modulate host cell function, and have evolved to sense host cell contact and to inject their substrates through a translocon pore in the host cell membrane. T3SS substrates contain an N-terminal signal sequence and often a chaperone-binding domain for cognate T3SS chaperones. These signals guide the substrates to the machine where substrates are unfolded and handed over to the secretion channel formed by the transmembrane domains of the export apparatus components and by the needle filament. Secretion itself is driven by the proton motive force across the bacterial inner membrane. The needle filament measures 20–150 nm in length and is crowned by a needle tip that mediates host-cell sensing. Secretion through T3SS is a highly regulated process with early, intermediate and late substrates. A strict secretion hierarchy is required to build an injectisome capable of reaching, sensing and penetrating the host cell membrane, before host cell-acting effector proteins are deployed. Here, we review the recent progress on elucidating the assembly, structure and function of T3SS injectisomes.

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“…Thus, a TMD that meets the hydrophobicity requirements for functioning both at the C terminus of an IMP and internally in a type II IMP may use different mechanisms to achieve efficient membrane insertion. Interestingly, a recent study describes a subset of effector proteins that are secreted via the type III and type IV secretion systems and yet have predicted TMDs of moderate hydrophobicity (37). It was shown that the predicted TMDs can indeed insert in the inner membrane when they are grafted into a polytopic IMP, suggesting a similar context dependency of TMD functioning.…”

Section: Discussionmentioning

confidence: 99%

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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Revealing the mechanisms of membrane protein export by virulence-associated bacterial secretion systems

Cited by 31 publications

References 51 publications

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Signal Recognition Particle Suppressor Screening Reveals the Regulation of Membrane Protein Targeting by the Translation Rate

Bacterial type III secretion systems: a complex device for the delivery of bacterial effector proteins into eukaryotic host cells

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

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