Insertase YidC Research Articles

Effect of the Membrane Insertase YidC on the Capacity of Lactococcus lactis to Secret Recombinant Proteins.

Lactococcus lactis is a crucial food-grade cell factory for secreting valuable peptides and proteins primarily via the Sec-dependent pathway. YidC, a membrane insertase, facilitates protein insertion into the lipid membrane for the translocation. However, the mechanistic details of how YidC affects protein secretion in L. lactis remain elusive. This study investigates the effects of deleting yidC1/yidC2 on L. lactis phenotypes and protein secretion. Compared to the original strain, deleting yidC2 significantly decreased the relative biomass, electroporation efficiency, and F-ATP activity by 25%, 47%, and 33%, respectively, and weakened growth and stress resistance, whereas deleting yidC1 had a minimal impact. The absence of either yidC1 or yidC2 reduced target proteins secretion. Meanwhile, there is a considerable alteration in the transcription levels of genes involved in the secretion pathway, with secY transcription increasing over 135-fold. Our results provide a theoretical foundation for further improving target protein secretion and investigating the YidC function.

Identifying the in vivo-induced antigenic genes is a strategy to develop DNA vaccine against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂)

Nocardia seriolae has been identified as the causative agent of fish nocardiosis, resulting in serious economic losses in aquaculture. With an aim to screen potential candidates for vaccine development against N. seriolae, the in vivo-induced genes of N. seriolae in hybrid snakehead (Channa maculate ♀ × Channa argus ♂) model were profiled via in vivo-induced antigen technology (IVIAT) in the present study, and 6 in vivo-induced genes were identified as follows: IS701 family transposase (is701), membrane protein insertase YidC (yidC), ergothioneine biosynthesis glutamate-cysteine ligase (egtA), molybdopterin respectively-dependent oxidoreductase (mol), phosphoketolase family protein (Ppl), hypothetical protein 6747 (hp6747). Additionally, the yidC was inserted into eukaryotic expression vector pcDNA3.1-myc-his-A to construct a DNA vaccine named as pcDNA-YidC to evaluate immunoprotection in hybrid snakehead after artificial challenge with N. serioale. Results showed that the transcription of yidC was detected in spleen, trunk kidney, muscle and liver in vaccinated fish, suggesting that this antigenic gene can be recombinantly expressed in fish. Meanwhile, indexes of humoral immunity were evaluated in the vaccinated fish through assessing specific-antibody IgM and serum enzyme activities, including lysozyme (LZM), superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (AKP). Quantitative real-time PCR analysis indicated that pcDNA-YidC DNA vaccine could notably enhance the expression of immune-related genes (CD4、CD8α、MHCIIα、TNFα、IL-1β and MHCIα) in 4 tissues (spleen, trunk kidney, muscle and liver) of the vaccinated fish. Finally, an immuno-protection with a relative survival rate of 65.71 % was displayed in vaccinated fish in comparison to the control groups. Taken together, these results indicate that pcDNA-YidC DNA vaccine could boost strong immune responses in hybrid snakehead and show preferably protective efficacy against N. seriolae, indicating that IVIAT is a helpful strategy to screen the highly immunogenic antigens for vaccine development against fish nocardiosis.

Key contributions of a glycolipid to membrane protein integration.

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

The insertase YidC chaperones the polytopic membrane protein MelB inserting and folding simultaneously from both termini

The insertion and folding of proteins into membranes is crucial for cell viability. Yet, the detailed contributions of insertases remain elusive. Here, we monitor how the insertase YidC guides the folding of the polytopic melibiose permease MelB into membranes. Invivo experiments using conditionally depleted E.coli strains show that MelB can insert in the absence of SecYEG if YidC resides in the cytoplasmic membrane. Invitro single-molecule force spectroscopy reveals that the MelB substrate itself forms two folding cores from which structural segments insert stepwise into the membrane. However, misfolding dominates, particularly in structural regions that interface the pseudo-symmetric α-helical domains of MelB. Here, YidC takes an important role in accelerating and chaperoning the stepwise insertion and folding process of both MelB folding cores. Our findings reveal a great flexibility of the chaperoning and insertase activity of YidC in the multifaceted folding processes of complex polytopic membrane proteins.

YidC as a potential antibiotic target

The membrane insertase YidC, is an essential bacterial component and functions in the folding and insertion of many membrane proteins during their biogenesis. It is a multispanning protein in the inner (cytoplasmic) membrane of Escherichia coli that binds its substrates in the “greasy slide” through hydrophobic interaction. The hydrophilic part of the substrate transiently localizes in the groove of YidC before it is translocated into the periplasm. The groove, which is flanked by the greasy slide, is within the center of the membrane, and provides a promising target for inhibitors that would block the insertase function of YidC. In addition, since the greasy slide is available for the binding of various substrates, it could also provide a binding site for inhibitory molecules. In this review we discuss in detail the structure and the mechanism of how YidC interacts not only with its substrates, but also with its partner proteins, the SecYEG translocase and the SRP signal recognition particle. Insight into the substrate binding to the YidC catalytic groove is presented. We wind up the review with the idea that the hydrophilic groove would be a potential site for drug binding and the feasibility of YidC-targeted drug development.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

Phytobacter diazotrophicus from Intestine of Caenorhabditis elegans Confers Colonization-Resistance against Bacillus nematocida Using Flagellin (FliC) as an Inhibition Factor.

Symbiotic microorganisms in the intestinal tract can influence the general fitness of their hosts and contribute to protecting them against invading pathogens. In this study, we obtained isolate Phytobacter diazotrophicus SCO41 from the gut of free-living nematode Caenorhabditis elegans that displayed strong colonization-resistance against invading biocontrol bacterium Bacillus nematocida B16. The colonization-resistance phenotype was found to be mediated by a 37-kDa extracellular protein that was identified as flagellin (FliC). With the help of genome information, the fliC gene was cloned and heterologously expressed in E. coli. It could be shown that the B. nematocida B16 grows in chains rather than in planktonic form in the presence of FliC. Scanning Electronic Microscopy results showed that protein FliC-treated B16 bacterial cells are thinner and longer than normal cells. Localization experiments confirmed that the protein FliC is localized in both the cytoplasm and the cell membrane of B16 strain, in the latter especially at the position of cell division. ZDOCK analysis showed that FliC could bind with serine/threonine protein kinase, membrane protein insertase YidC and redox membrane protein CydB. It was inferred that FliC interferes with cell division of B. nematocidal B16, therefore inhibiting its colonization of C. elegans intestines in vivo. The isolation of P. diazotrophicus as part of the gut microbiome of C. elegans not only provides interesting insights about the lifestyle of this nitrogen-fixing bacterium, but also reveals how the composition of the natural gut microbiota of nematodes can affect biological control efforts by protecting the host from its natural enemies.

A bacterial glycolipid essential for membrane protein integration.

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.

The role of the N-terminal amphipathic helix in bacterial YidC: Insights from functional studies, the crystal structure and molecular dynamics simulations

The evolutionary conserved YidC is a unique dual-function membrane protein that adopts insertase and chaperone conformations. The N-terminal helix of Escherichia coli YidC functions as an uncleaved signal sequence and is important for membrane insertion and interaction with the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the N-terminal amphipathic helix (N-AH) (PDB ID: 6Y86). Molecular dynamics simulations show that N-AH lies on the periplasmic side of the membrane bilayer forming an angle of about 15° with the membrane surface. Our functional studies suggest a role of N-AH for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that N-AH acts as a membrane recognition helix for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of N-AH and the other structural elements.

Monitoring the binding and insertion of a single transmembrane protein by an insertase

Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.

Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion

Significance Membrane proteins are inserted into the phospholipid bilayer through a lateral gate in the translocon, SecYEG in bacteria, which is expected to be closed in the resting state. Here, we use single-molecule FRET to study the translocon dynamics on timescales ranging from submilliseconds to seconds. We show that the lateral gate is highly dynamic, fluctuating through a continuum of states from open to closed. The insertase YidC facilitates the insertion of transmembrane helices by shifting the fluctuations toward more open conformations. Spontaneous fluctuations allow the gate to rapidly release newly synthesized transmembrane segments into the phospholipid bilayer during ongoing translation. The results highlight the important role of rapid spontaneous fluctuations during the key step in the biogenesis of inner-membrane proteins.

Molecular communication of the membrane insertase YidC with translocase SecYEG affects client proteins

The membrane insertase YidC inserts newly synthesized proteins by its hydrophobic slide consisting of the two transmembrane (TM) segments TM3 and TM5. Mutations in this part of the protein affect the insertion of the client proteins. We show here that a quintuple mutation, termed YidC-5S, inhibits the insertion of the subunit a of the FoF1 ATP synthase but has no effect on the insertion of the Sec-independent M13 procoat protein and the C-tail protein SciP. Further investigations show that the interaction of YidC-5S with SecY is inhibited. The purified and fluorescently labeled YidC-5S did not approach SecYEG when both were co-reconstituted in proteoliposomes in contrast to the co-reconstituted YidC wild type. These results suggest that TM3 and TM5 are involved in the formation of a common YidC-SecYEG complex that is required for the insertion of Sec/YidC-dependent client proteins.

Investigating YidC-assisted Folding Pathways of Different Alpha-Helical Membrane Proteins

Insertion and folding of multi-spanning alpha-helical membrane proteins into the inner membrane of bacteria are crucial steps during protein biogenesis. These processes are assisted by a universally conserved machinery, the YidC insertase. It has been recently shown that YidC initiates folding of the lactose permease LacY by inserting a single structural segment into the membrane. Further folding of the LacY polypeptide occurs along variable trajectories until it reaches its native structure. However, to understand how YidC assists the insertion and folding of other membrane proteins, we first mechanically extract alpha-helical proteins, namely BetT3 and MelB, from a membrane using single-molecule force spectroscopy and record their characteristic unfolding spectra.

Probing the Impact of Lipid Composition on Structure and Dynamics of E. coli Protein Insertase YidC

Lipid-protein interactions have been shown to play an important regulatory role in the function of membrane proteins. Nevertheless, due to the semi-liquid nature and heterogeneity of biological membranes, dissecting the details of such interactions at high resolutions continues to pose a major challenge to experimental biophysical techniques. Computational techniques such as molecular dynamics (MD) offer an alternative approach with both temporally and spatially high resolutions. Here we report an extensive set of MD simulations investigating the structure and dynamics of the E. coli inner membrane protein YidC (RCSB: 6AL2), a small protein insertase, as a model protein to probe the differences in protein-lipid interactions in three membrane compositions. The lipid bilayers used in our study include: (1) pure POPE membranes, which have been often used to represent bacterial membranes in a simple mono-lipidic bilayer; (2) a 3:1 mixture of POPE:POPG, taking into account one of the important anionic lipids of bacterial membranes, roughly capturing the headgroup diversity found in the model organism; and (3) a more accurate model for the inner membrane of E. coli has been developed, called Top6, which contains an approximately 3:1 ratio of PG:PE headgroups, and more importantly several lipids with cyclopropane moieties and variable acyl tail lengths which give rise to better agreement with experimentally derived membrane features such as thickness and deuterium order parameters and lipid abundances. Several independent simulation replicas were generated for each lipid composition and each simulated for at least 500 ns using atomistic MD. Analysis of the results of this ensemble of simulations illustrate differential lipid-protein interactions in different membrane contexts, highlighting that biologically-accurate membranes are crucial to the study of protein-membrane systems with MD.

Xanthophyll carotenoids stabilise the association of cyanobacterial chlorophyll synthase with the LHC-like protein HliD

Chlorophyll synthase (ChlG) catalyses a terminal reaction in the chlorophyll biosynthesis pathway, attachment of phytol or geranylgeraniol to the C17 propionate of chlorophyllide. Cyanobacterial ChlG forms a stable complex with high light-inducible protein D (HliD), a small single-helix protein homologous to the third transmembrane helix of plant light-harvesting complexes (LHCs). The ChlG-HliD assembly binds chlorophyll, β-carotene, zeaxanthin and myxoxanthophyll and associates with the YidC insertase, most likely to facilitate incorporation of chlorophyll into translated photosystem apoproteins. HliD independently coordinates chlorophyll and β-carotene but the role of the xanthophylls, which appear to be exclusive to the core ChlG-HliD assembly, is unclear. Here we generated mutants of Synechocystis sp. PCC 6803 lacking specific combinations of carotenoids or HliD in a background with FLAG- or His-tagged ChlG. Immunoprecipitation experiments and analysis of isolated membranes demonstrate that the absence of zeaxanthin and myxoxanthophyll significantly weakens the interaction between HliD and ChlG. ChlG alone does not bind carotenoids and accumulation of the chlorophyllide substrate in the absence of xanthophylls indicates that activity/stability of the 'naked' enzyme is perturbed. In contrast, the interaction of HliD with a second partner, the photosystem II assembly factor Ycf39, is preserved in the absence of xanthophylls. We propose that xanthophylls are required for the stable association of ChlG and HliD, acting as a 'molecular glue' at the lateral transmembrane interface between these proteins; roles for zeaxanthin and myxoxanthophyll in ChlG-HliD complexation are discussed, as well as the possible presence of similar complexes between LHC-like proteins and chlorophyll biosynthesis enzymes in plants.

Polarity/charge as a determinant of translocase requirements for membrane protein insertion

The YidC insertase of Escherichia coli inserts membrane proteins with small periplasmic loops (~20 residues). However, it has difficulty transporting loops that contain positively charged residues compared to negatively charged residues and, as a result, increasing the positive charge has an increased requirement for the Sec machinery as compared to negatively charged loops (Zhu et al., 2013; Soman et al., 2014). This suggested that the polarity and charge of the periplasmic regions of membrane proteins determine the YidC and Sec translocase requirements for insertion. Here we tested this polarity/charge hypothesis by showing that insertion of our model substrate protein procoat-Lep can become YidC/Sec dependent when the periplasmic loop was converted to highly polar even in the absence of any charged residues. Moreover, adding a number of hydrophobic amino acids to a highly polar loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent membrane proteins. We also demonstrate that the length of the procoat-Lep loop is indeed a determinant for Sec-dependence by inserting alanine residues that do not markedly change the overall hydrophilicity of the periplasmic loop. Taken together, the results support the polarity/charge hypothesis as a determinant for the translocase requirement for procoat insertion.

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

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 27-amino-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.

Structure of the ER membrane complex, a transmembrane-domain insertase.

The ER membrane complex (EMC) cooperates with the Sec61 translocon to co-translationally insert a transmembrane helix (TMH) of many multi-pass integral membrane proteins into the ER membrane, and it is also responsible for inserting the TMH of some tail-anchored proteins 1–3. How EMC accomplishes this feat has been unclear. Here we report the first cryo-EM structure of the eukaryotic EMC. We found that the Saccharomyces cerevisiae EMC contains eight subunits (Emc1–6, 7, and 10); has a large lumenal region and a smaller cytosolic region; and has a transmembrane region formed by Emc4, 5, and 6 plus the transmembrane domains (TMDs) of Emc1 and 3. We identified a 5-TMH fold centered around Emc3 that resembles the prokaryotic insertase YidC and that delineates a largely hydrophilic client pocket. The TMD of Emc4 tilts away from the main transmembrane region of EMC and is partially mobile. Mutational studies demonstrated that Emc4 flexibility and the hydrophilicity of the client pocket are required for EMC function. The EMC structure reveals a remarkable evolutionary conservation with the prokaryotic insertases 4,5; suggests a similar mechanism of TMH insertion; and provides a framework for detailed understanding of membrane insertion for numerous eukaryotic integral membrane proteins and tail-anchored proteins.

In-Silico Proteomic Exploratory Quest: Crafting T-Cell Epitope Vaccine Against Whipple's Disease.

Whipple’s disease is one of the rare maladies in terms of spread but very fatal one as it is linked with many disorders (like Gastroenteritis, Endocarditis etc.). Also, current regimens include less effective drugs which require long duration follows up. This exploratory study was conducted to commence the investigation for crafting multi target epitope vaccine against its bacterial pathogen Tropheryma whipplei. The modern bioinformatics tools like VaxiJen, NETMHCII PAN 3.2, ALLERGEN-FP, PATCH-DOCK, TOXIC-PRED, MHCPRED and IEDB were deployed, which makes the study more intensive in analyzing proteome of T. whipplei as these methods are based on robust result generating statistical algorithms ANN, HMM, and ML. This Immuno-Informatics approach leads us in the prediction of two epitopes: VLMVSAFPL and IRYLAALHL interacting with 4 and 6 HLA DRB1 alleles of MHC Class II respectively. VLMVSAFPL epitope is a part of DNA-directed RNA polymerase subunit beta, and IRYLAALHL epitope is a part of membranous protein insertase YidC of this bacterium. Molecular-Docking and Molecular-Simulation analysis yields the perfect interaction based on Atomic contact energy, binding scores along with RMSD values (0 to 1.5 Ǻ) in selection zone. The IEDB (Immune epitope database) population coverage analysis exhibits satisfactory relevance with respect to world population.

Cardiolipin is required in vivo for the stability of bacterial translocon and optimal membrane protein translocation and insertion

Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane β-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomerization properties of SecA were strongly compromised while the levels of individual SecYEG translocon components, SecA and insertase YidC were largely unaffected. These results demonstrate that CL is required in vivo for the stability of the bacterial translocon and its efficient function in co-translational insertion into and translocation across the inner membrane of E. coli.