SecA is required for the insertion of inner membrane proteins targeted by the Escherichia coli signal recognition particle

Recent work has demonstrated that the signal recognition particle (SRP) is required for the efficient insertion of many proteins into the Escherichia coli inner membrane (IM). Based on an analogy to eukaryotic SRP, it is likely that bacterial SRP binds to inner membrane proteins (IMPs) co-translationally and then targets them to protein transport channels ("translocons"). Here we present evidence that SecA, which has previously been shown to facilitate the export of proteins targeted in a post-translational fashion, is also required for the membrane insertion of proteins targeted by SRP. The introduction of SecA mutations into strains that have modest SRP deficiencies produced a synthetic lethal effect, suggesting that SecA and SRP might function in the same biochemical pathway. Consistent with this explanation, depletion of SecA by inactivating a temperature-sensitive amber suppressor in a secAam strain completely blocked the membrane insertion of AcrB, a protein that is targeted by SRP. In the absence of substantial SecA, pulse-labeled AcrB was retained in the cytoplasm even after a prolonged chase period and was eventually degraded. Although protein export was also severely impaired by SecA depletion, the observation that more than 20% of the OmpA molecules were translocated properly showed that translocons were still active. Taken together, these results imply that SecA plays a much broader role in the transport of proteins across the E. coli IM than has been previously recognized.     

Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway

In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs). At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer. The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer. It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied. These model proteins do not include lipoproteins. Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach. The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase. Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.     

Export of beta-lactamase is independent of the signal recognition particle

In Escherichia coli, three different types of proteins engage the SecY translocon of the inner bacterial membrane for translocation or insertion: 1) polytopic membrane proteins that prior to their insertion into the membrane are targeted to the translocon using the bacterial signal recognition particle (SRP) and its receptor; 2) secretory proteins that are targeted to and translocated across the SecY translocon in a SecA- and SecB-dependent reaction; and 3) membrane proteins with large periplasmic domains, requiring SRP for targeting and SecA for the translocation of the periplasmic moiety. In addition to its role as a targeting device for membrane proteins, a function of the bacterial SRP in the export of SecB-independent secretory proteins has also been postulated. In particular, beta-lactamase, a hydrolytic enzyme responsible for cleavage of the beta-lactam ring containing antibiotics, is considered to be recognized and targeted by SRP. To examine the role of the SRP pathway in beta-lactamase targeting and export, we performed a detailed in vitro analysis. Chemical cross-linking and membrane binding assays did not reveal any significant interaction between SRP and beta-lactamase nascent chains. More importantly, membrane vesicles prepared from mutants lacking a functional SRP pathway did block the integration of SRP-dependent membrane proteins but supported the export of beta-lactamase in the same way as that of the SRP-independent protein OmpA. These data demonstrate that in contrast to previous results, the bacterial SRP is not involved in the export of beta-lactamase and further suggest that secretory proteins of Gram-negative bacteria in general are not substrates of SRP.     

Reprogramming chaperone pathways to improve membrane protein expression in Escherichia coli

Because membrane proteins are difficult to express, our understanding of their structure and function is lagging. In Escherichia coli, α-helical membrane protein biogenesis usually involves binding of a nascent transmembrane segment (TMS) by the signal recognition particle (SRP), delivery of the SRP-ribosome nascent chain complexes (RNC) to FtsY, a protein that serves as SRP receptor and docks to the SecYEG translocon, cotranslational insertion of the growing chain into the translocon, and lateral transfer, packing and folding of TMS in the lipid bilayer in a process that may involve chaperone YidC. Here, we explored the feasibility of reprogramming this pathway to improve the production of recombinant membrane proteins in exponentially growing E. coli with a focus on: (i) eliminating competition between SRP and chaperone trigger factor (TF) at the ribosome through gene deletion; (ii) improving RNC delivery to the inner membrane via SRP overexpression; and (iii) promoting substrate insertion and folding in the lipid bilayer by increasing YidC levels. Using a bitopic histidine kinase and two heptahelical rhodopsins as model systems, we show that the use of TF-deficient cells improves the yields of membrane-integrated material threefold to sevenfold relative to the wild type, and that whereas YidC coexpression is beneficial to the production of polytopic proteins, higher levels of SRP have the opposite effect. The implications of our results on the interplay of TF, SRP, YidC, and SecYEG in membrane protein biogenesis are discussed.     

Insertion of bitopic membrane proteins into the inner membrane of mitochondria involves an export step from the matrix

The mitochondrial inner membrane contains a large number of polytopic proteins that are derived from prokaryotic ancestors of mitochondria. Little is known about the intramitochondrial sorting of these proteins. We chose two proteins of known topology as examples to study the pathway of insertion into the inner membrane; Mrs2 and Yta10 are bitopic proteins that expose negatively charged loops of different complexity into the intermembrane space. Here we show that both Mrs2 and Yta10 transiently accumulate as sorting intermediates in the matrix before they integrate into the inner membrane. The sorting pathway of both proteins can be separated into two sequential reactions: (i) import into the matrix and (ii) insertion from the matrix into the inner membrane. The latter process was found to depend on the membrane potential and, in this respect, is similar to the insertion of membrane proteins in bacteria. A comparison of the charge distribution of intermembrane space loops in a variety of mitochondrial inner membrane proteins suggests that this mode of "conservative sorting" might be the typical insertion route for polytopic inner membrane proteins that originated from bacterial ancestors.     

Topogenesis of mitochondrial inner membrane uncoupling protein. Rerouting transmembrane segments to the soluble matrix compartment

Brown adipose tissue uncoupling protein (UCP), an integral polytopic protein of the mitochondrial inner membrane, is composed of at least six transmembrane segments whose net hydrophobic character derives from paired amphiphilic helices. The protein is synthesized in the cytoplasm as a polypeptide (307 amino acids) lacking a cleavable targeting (signal) peptide. Deletion mutagenesis and fusion protein constructions revealed the existence of at least two import signals: one lying between UCP precursor amino acids 13-105 and the other downstream of position 101. The former resulted in both targeting and membrane insertion of a fusion protein, whereas the latter targeted UCP 102-307 into the organelle but failed to result in membrane insertion. When a strong matrix-targeting signal derived from precarbamoyl phosphate synthetase was fused to UCP amino acids 169-307 or 52-307 (containing three and five transmembrane domains, respectively), the fusion proteins were efficiently imported to the soluble matrix compartment where correct signal cleavage took place. We suggest that assembly of UCP into the inner membrane follows a coordinate insertion pathway for integration and may use more than one signal sequence to achieve this. In this respect, it might share certain mechanistic features with the insertion of polytopic proteins into the endoplasmic reticulum. The data also suggest, however, that integration of the amino-terminal third of UCP into the inner membrane may be required to help or enhance insertion of the remaining UCP transmembrane domains.     

Rational design of a fusion partner for membrane protein expression in E. coli

We have designed a novel protein fusion partner (P8CBD) to utilize the co‐translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP‐dependence was demonstrated by analyzing the membrane translocation of P8CBD‐PhoA fusion proteins in wt and SRP‐ffh77 mutant cells. We also demonstrate that the P8CBD N‐terminal fusion partner promotes over‐expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over‐expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane‐associated protein.     

Flexibility in targeting and insertion during bacterial membrane protein biogenesis

The biogenesis of Escherichia coli inner membrane proteins (IMPs) is assisted by targeting and insertion factors such as the signal recognition particle (SRP), the Sec-translocon and YidC with translocation of (large) periplasmic domains energized by SecA and the proton motive force (pmf). The use of these factors and forces is probably primarily determined by specific structural features of an IMP. To analyze these features we have engineered a set of model IMPs based on endogenous E. coli IMPs known to follow distinct targeting and insertion pathways. The modified model IMPs were analyzed for altered routing using an in vivo protease mapping approach. The data suggest a facultative use of different combinations of factors.     

Physiological basis for conservation of the signal recognition particle targeting pathway in Escherichia coli

The Escherichia coli signal recognition particle (SRP) is a ribonucleoprotein complex that targets nascent inner membrane proteins (IMPs) to transport sites in the inner membrane (IM). Since SRP depletion only partially inhibits IMP insertion under some growth conditions, however, it is not clear why the particle is absolutely essential for viability. Insights into this question emerged from experiments in which we analyzed the physiological consequences of reducing the intracellular concentration of SRP below the wild-type level. We found that even moderate SRP deficiencies that have little effect on cell growth led to the induction of a heat shock response. Genetic manipulations that suppress the heat shock response were lethal in SRP-deficient cells, indicating that the elevated synthesis of heat shock proteins plays an important role in maintaining cell viability. Although it is conceivable that the heat shock response serves to increase the capacity of cells to target IMPs via chaperone-based mechanisms, SRP-deficient cells did not show an increased dependence on either GroEL or DnaK. By contrast, the heat shock-regulated proteases Lon and ClpQ became essential for viability when SRP levels were reduced. These results suggest that the heat shock response protects SRP-deficient cells by increasing their capacity to degrade mislocalized IMPs. Consistent with this notion, a model IMP that was mislocalized in the cytoplasm as the result of SRP depletion appeared to be more stable in a Deltalon DeltaclpQ strain than in control cells. Taken together, the data provide direct evidence that SRP is essential in E. coli and possibly conserved throughout prokaryotic evolution as well partly because efficient IMP targeting prevents a toxic accumulation of aggregated proteins in the cytoplasm.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Function of a chloroplast SRP in thylakoid protein export.

Protein export systems derived from prokaryotes are used to transport proteins into or across the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoid membrane. Signal recognition particle (SRP) and its receptor are essential components used exclusively for cotranslational export of endomembrane and secretory proteins to the endoplasmic reticulum in eukaryotes and export of polytopic membrane proteins to the cytoplasmic membrane in prokaryotes. An organellar SRP in chloroplasts (cpSRP) participates in cotranslational targeting of chloroplast synthesized integral thylakoid proteins. Remarkably, cpSRP is also used to posttranslationally localize a subset of nuclear encoded thylakoid proteins. Recent work has begun to reveal the basis for cpSRP's unique ability to function in co- and posttranslational protein localization, yet much is left to question. This review will attempt to highlight these advances and will also focus on the role of other soluble and membrane components that are part of this novel organellar SRP targeting pathway.     

Discrimination between SRP- and SecA/SecB-dependent substrates involves selective recognition of nascent chains by SRP and trigger factor

Besides SecA and SecB, Escherichia coli cells possess a signal recognition particle (SRP) to target exported proteins to the SecY translocon. Using chemical and site-specific cross-linking in vitro, we show that SRP recognizes the first signal anchor sequence of a polytopic membrane protein (MtlA) resulting in cotranslational targeting of MtlA to SecY and phospholipids of the plasma membrane. In contrast, a possible interaction of SRP with the secretory protein pOmpA is prevented by the association of trigger factor with nascent pOmpA. Trigger factor also prevents SecA from binding to the first 125 amino acids of pOmpA when they are still associated with the ribosome. Under no experimental conditions was SecA found to interact with MtlA. Likewise, virtually no binding of trigger factor to ribosome-bound MtlA occurs even in the complete absence of SRP. Collectively, our results indicate that at the stage of nascent polypeptides, polytopic membrane proteins are selected by SRP for co-translational membrane targeting, whereas secretory proteins are directed into the SecA/SecB-mediated post-translational targeting pathway by means of their preferential recognition by trigger factor.     

F(1)F(0) ATP synthase subunit c is targeted by the SRP to YidC in the E. coli inner membrane

Escherichia coli inner membrane proteins (IMPs) use different pathways for targeting and membrane integration. We have examined the biogenesis of the F1F0 ATP synthase subunit c, a small double spanning IMP, using complementary in vivo and in vitro approaches. The data suggest that F0c is targeted by the SRP to the membrane, where it inserts at YidC in a Sec-independent mechanism. F0c appears to be the first natural substrate of this novel pathway.     

Down-regulation of the trypanosomatid signal recognition particle affects the biogenesis of polytopic membrane proteins but not of signal peptide-containing proteins

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.     

Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.     

SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein.

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.     

Targeting and insertion of heterologous membrane proteins in E. coli

Membrane targeting and insertion of the archaeal Halobacter halobium proton pump bacterioopsin (Bop) and the human melanocortin 4 receptor (MC(4)R) were studied in vitro, using E. coli components for protein synthesis, membrane targeting and insertion. These heterologous proteins are targeted to E. coli membranes in a signal recognition particle (SRP) dependent manner and inserted into the membrane co-translationally. Furthermore, we demonstrate that nascent chains of Bop and MC(4)R first interact with SecY and then with YidC as they move through the translocon. Our results suggest that the initial stages of membrane targeting and insertion of heterologous proteins in E. coli proceed by the pathway used for native E. coli membrane proteins. No significant pausing of protein elongation was observed in the presence of E. coli SRP, in line with the suggestion that translational arrest requires an Alu domain, which is absent in SRP from E. coli.     

Dissecting the translocase and integrase functions of the Escherichia coli SecYEG translocon

Recent evidence suggests that in Escherichia coli, SecA/SecB and signal recognition particle (SRP) are constituents of two different pathways targeting secretory and inner membrane proteins to the SecYEG translocon of the plasma membrane. We now show that a secY mutation, which compromises a functional SecY-SecA interaction, does not impair the SRP-mediated integration of polytopic inner membrane proteins. Furthermore, under conditions in which the translocation of secretory proteins is strictly dependent on SecG for assisting SecA, the absence of SecG still allows polytopic membrane proteins to integrate at the wild-type level. These results indicate that SRP-dependent integration and SecA/SecB-mediated translocation do not only represent two independent protein delivery systems, but also remain mechanistically distinct processes even at the level of the membrane where they engage different domains of SecY and different components of the translocon. In addition, the experimental setup used here enabled us to demonstrate that SRP-dependent integration of a multispanning protein into membrane vesicles leads to a biologically active enzyme.     

Contact-dependent growth inhibition requires the essential outer membrane protein BamA (YaeT) as the receptor and the inner membrane transport protein AcrB

Contact-dependent growth inhibition (CDI) is a phenomenon by which bacterial cell growth is regulated by direct cell-to-cell contact via the CdiA/CdiB two-partner secretion system. Characterization of mutants resistant to CDI allowed us to identify BamA (YaeT) as the outer membrane receptor for CDI and AcrB as a potential downstream target. Notably, both BamA and AcrB are part of distinct multi-component machines. The Bam machine assembles outer membrane beta-barrel proteins into the outer membrane and the Acr machine exports small molecules into the extracellular milieu. We discovered that a mutation that reduces expression of BamA decreased binding of CDI+ inhibitor cells, measured by flow cytometry with fluorescently labelled bacteria. In addition, alpha-BamA antibodies, which recognized extracellular epitopes of BamA based on immunofluorescence, specifically blocked inhibitor-target cells binding and CDI. A second class of CDI-resistant mutants identified carried null mutations in the acrB gene. AcrB is an inner membrane component of a multidrug efflux pump that normally forms a cell envelope-spanning complex with the membrane fusion protein AcrA and the outer membrane protein TolC. Strikingly, the requirement for the BamA and AcrB proteins in CDI is independent of their multi-component machines, and thus their role in the CDI pathway may reflect novel, import-related functions.