Biogenesis of inner membrane proteins in Escherichia coli

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.     

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.     

The signal recognition particle-targeting pathway does not necessarily deliver proteins to the sec-translocase in Escherichia coli.

ProW is an Escherichia coli inner membrane protein that consists of a 100-residue-long periplasmic N-terminal tail (N-tail) followed by seven closely spaced transmembrane segments. N-tail translocation presumably proceeds in a C-to-N-terminal direction and represents a poorly understood aspect of membrane protein biogenesis. Here, using an in vivo depletion approach, we show that N-tail translocation in a ProW derivative comprising the N-tail and the first transmembrane segment fused to the globular P2 domain of leader peptidase depends both on the bacterial signal recognition particle (SRP) and the Sec-translocase. Surprisingly, however, a deletion construct with only one transmembrane segment downstream of the N-tail can assemble properly even under severe depletion of SecE, a central component of the Sec-translocase, but not under SRP-depletion conditions. To our knowledge, this is the first demonstration that the SRP-targeting pathway does not necessarily deliver SRP-dependent inner membrane proteins to the Sec-translocase. The data further suggest that N-tail translocation can proceed in the absence of a functional Sec-translocase.     

YidC is involved in the biogenesis of anaerobic respiratory complexes in the inner membrane of Escherichia coli

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of this family have all been implicated in membrane protein biogenesis of aerobic respiratory and energy-transducing proteins. YidC is essential for the insertion of subunit c of the F(1)F(0)-ATP synthase and subunit a of cytochrome o oxidase. The aim of this study was to investigate whether YidC plays a role during anaerobic growth of Escherichia coli, specifically when either nitrate or fumarate are used as terminal electron acceptors or under fermentative conditions. The effect of YidC depletion on the growth, enzyme activities, and protein levels in the inner membrane was determined. YidC is essential for all anaerobic growth conditions tested, and this is not because of the decreased levels of F(1)F(0)-ATP synthase in the inner membrane only. The results suggest a role for YidC in the membrane biogenesis of integral membrane parts of the anaerobic respiratory chain.     

Saccharomyces cerevisiae Cox18 complements the essential Sec-independent function of Escherichia coli YidC

Members of the YidC/Oxa1/Alb3 protein family function in the biogenesis of membrane proteins in bacteria, mitochondria and chloroplasts. In Escherichia coli, YidC plays a key role in the integration and assembly of many inner membrane proteins. Interestingly, YidC functions both in concert with the Sec-translocon and as a separate insertase independent of the translocon. Mitochondria of higher eukaryotes contain two distant homologues of YidC: Oxa1 and Cox18/Oxa2. Oxa1 is required for the insertion of membrane proteins into the mitochondrial inner membrane. Cox18/Oxa2 plays a poorly defined role in the biogenesis of the cytochrome c oxidase complex. Employing a genetic complementation approach by expressing the conserved region of yeast Cox18 in E. coli, we show here that Cox18 is able to complement the essential Sec-independent function of YidC. This identifies Cox18 as a bona fide member of the YidC/Oxa1/Alb3 family.     

Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs.     

Differential effect of YidC depletion on the membrane proteome of Escherichia coli under aerobic and anaerobic growth conditions

YidC of Escherichia coli belongs to the evolutionarily conserved Oxa1/Alb3/YidC family. Members of the family have all been implicated in membrane protein biogenesis of respiratory and energy transducing proteins. The number of proteins identified thus far to require YidC for their membrane biogenesis remains limited and the identification of new substrates may allow the elucidation of properties that define the YidC specificity. To this end we investigated changes in the membrane proteome of E. coli upon YidC depletion using metabolic labeling of proteins with ¹⁵N/¹⁴N combined with a MS‐centered proteomics approach and compared the effects of YidC depletion under aerobic and anaerobic growth conditions. We found that YidC depletion resulted in protein aggregation/misfolding in the cytoplasm as well as in the inner membrane of E. coli. A dramatic increase was observed in the chaperone‐mediated stress response upon YidC depletion and this response was limited to aerobically grown cells. A number of transporter proteins were identified as possible candidates for the YidC‐dependent insertion and/or folding pathway. These included the small metal ion transporter CorA, numerous ABC transporters, as well as the MFS transporters KgtP and ProP, providing a new subset of proteins potentially requiring YidC for membrane biogenesis.     

Versatility of inner membrane protein biogenesis in Escherichia coli

To further our understanding of inner membrane protein (IMP) biogenesis in Escherichia coli, we have accomplished the widest in vivo IMP assembly screen so far. The biogenesis of a set of model IMPs covering most IMP structures possible has been studied in a variety of signal recognition particle (SRP), Sec and YidC mutant strains. We show that the assembly of the complete set of model IMPs is assisted (i.e. requires the aid of proteinaceous factors), and that the requirements for assembly of the model IMPs into the inner membrane differ significantly from each other. This indicates that IMP assembly is much more versatile than previously thought.     

Consequences of depletion of the signal recognition particle in Escherichia coli

Thus far, the role of the Escherichia coli signal recognition particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a σ(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.     

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.     

Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli.

Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism. It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion. During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion). We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis. This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate. Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules. These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.     

Characterization of the consequences of YidC depletion on the inner membrane proteome of E. coli using 2D blue native/SDS-PAGE

In the bacterium Escherichia coli, the essential inner membrane protein (IMP) YidC assists in the biogenesis of IMPs and IMP complexes. Our current ideas about the function of YidC are based on targeted approaches using only a handful of model IMPs. Proteome-wide approaches are required to further our understanding of the significance of YidC and to find new YidC substrates. Here, using two-dimensional blue native/SDS-PAGE methodology that is suitable for comparative analysis, we have characterized the consequences of YidC depletion for the steady-state levels and oligomeric state of the constituents of the inner membrane proteome. Our analysis showed that (i) YidC depletion reduces the levels of a variety of complexes without changing their composition, (ii) the levels of IMPs containing only soluble domains smaller than 100 amino acids are likely to be reduced upon YidC depletion, whereas the levels of IMPs with at least one soluble domain larger than 100 amino acids do not, and (iii) the levels of a number of proteins with established or putative chaperone activity (HflC, HflK, PpiD, OppA, GroEL and DnaK) are strongly increased in the inner membrane fraction upon YidC depletion. In the absence of YidC, these proteins may assist the folding of sizeable soluble domains of IMPs, thereby supporting their folding and oligomeric assembly. In conclusion, our analysis identifies many new IMPs/IMP complexes that depend on YidC for their biogenesis, responses that accompany depletion of YidC and an IMP characteristic that is associated with YidC dependence.     

Folding of a bacterial outer membrane protein during passage through the periplasm.

The transport of bacterial outer membrane proteins to their destination might be either a one-step process via the contact zones between the inner and outer membrane or a two-step process, implicating a periplasmic intermediate that inserts into the membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the outer membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the outer membrane.     

Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli

The Sec translocon is a protein-conducting channel that allows polypeptides to be transferred across or integrated into a membrane. Although protein translocation and insertion in Escherichia coli have been studied using only a small set of specific model substrates, it is generally assumed that most secretory proteins and inner membrane proteins use the Sec translocon. Therefore, we have studied the role of the Sec translocon using subproteome analysis of cells depleted of the essential translocon component SecE. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and extensive immunoblotting. The analysis showed that upon SecE depletion (i) secretory proteins aggregated in the cytoplasm and the cytoplasmic sigma(32) stress response was induced, (ii) the accumulation of outer membrane proteins was reduced, with the exception of OmpA, Pal, and FadL, and (iii) the accumulation of a surprisingly large number of inner membrane proteins appeared to be unaffected or increased. These proteins lacked large translocated domains and/or consisted of only one or two transmembrane segments. Our study suggests that several secretory and inner membrane proteins can use Sec translocon-independent pathways or have superior access to the remaining Sec translocons present in SecE-depleted cells.     

Structure and function of the bacterial AAA protease FtsH

Proteolysis of regulatory proteins or key enzymes of biosynthetic pathways is a universal mechanism to rapidly adjust the cellular proteome to particular environmental needs. Among the five energy-dependent AAA(+) proteases in Escherichia coli, FtsH is the only essential protease. Moreover, FtsH is unique owing to its anchoring to the inner membrane. This review describes the structural and functional properties of FtsH. With regard to its role in cellular quality control and regulatory circuits, cytoplasmic and membrane substrates of the FtsH protease are depicted and mechanisms of FtsH-dependent proteolysis are discussed.     

Genome-wide identification of the subcellular localization of the Escherichia coli B proteome using experimental and computational methods

Escherichia coli K-12 and B strains have most widely been employed for scientific studies as well as industrial applications. Recently, the complete genome sequences of two representative descendants of E. coli B strains, REL606 and BL21(DE3), have been determined. Here, we report the subproteome reference maps of E. coli B REL606 by analyzing cytoplasmic, periplasmic, inner and outer membrane, and extracellular proteomes based on the genome information using experimental and computational approaches. Among the total of 3487 spots, 651 proteins including 410 non-redundant proteins were identified and characterized by 2-DE and LC-MS/MS; they include 440 cytoplasmic, 45 periplasmic, 50 inner membrane, 61 outer membrane, and 55 extracellular proteins. In addition, subcellular localizations of all 4205 ORFs of E. coli B were predicted by combined computational prediction methods. The subcellular localizations of 1812 (43.09%) proteins of currently unknown function were newly assigned. The results of computational prediction were also compared with the experimental results, showing that overall precision and recall were 92.16 and 92.16%, respectively. This work represents the most comprehensive analyses of the subproteomes of E. coli B, and will be useful as a reference for proteome profiling studies under various conditions. The complete proteome data are available online (http://ecolib.kaist.ac.kr).     

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.     

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.     

Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili

Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.