Insertase YidC Research Articles

Identification of YidC Residues That Define Interactions with the Sec Apparatus

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.

Single-Molecule Studies of Bacterial Protein Translocation

In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of eukaryotes. The Sec system also facilitates the membrane insertion of integral membrane proteins, an essential step along their folding pathway. In bacteria, the Sec system consists of the protein-conducting channel (SecYEG) that associates with soluble components, such as the motor protein SecA or translating ribosomes, and with integral membrane proteins, such as the heterotrimeric complex termed SecDFyajC and the YidC insertase. Over the past three decades, biochemical and structural studies have provided a comprehensive view of protein translocation, but the exact mechanistic details of this process remain to be resolved. For a number of other biomolecular systems, single-molecule biophysical analysis has efficiently complemented the conventional biochemical studies conducted in bulk, with high-sensitivity measurements probing the structure and dynamics of individual molecules in vitro and in vivo. Here, we review recent advances in studies of protein translocation employing single-molecule techniques with the aim of resolving molecular mechanisms, thereby providing a new and detailed view of the process.

Protein translocation across the inner membrane of Gram-negative bacteria: the Sec and Tat dependent protein transport pathways

Gram negative bacteria possess a large variety of protein transport systems, by which proteins that are synthesised in the cytosol are exported to destinations in the cell envelope or entirely secreted into the extracellular environment. The inner membrane (IM) contains three major transport systems for the translocation and insertion of signal sequence containing proteins: the Sec translocon, the YidC insertase, and the Tat system. The heterotrimeric SecYEG translocon forms a narrow channel in the membrane that serves a dual function; it allows the translocation of unfolded proteins across the pore and the integration of α-helical proteins into the IM. The YidC insertase is a multi-spanning membrane protein that cooperates with the SecYEG translocon during the integration of membrane proteins but also functions as an independent insertase. Depending upon the type of protein cargo that needs to be transported, the Signal Recognition Particle (SRP), the SRP receptor, SecA and chaperones are required to coordinate translation with transport and to target and energise the different transport systems. The Tat system consists of three membrane proteins (TatA, TatB and TatC) which in a still unknown manner accomplish the transmembrane passage of completely folded proteins and protein complexes.

Real Time Observation of Single Membrane Protein Insertion Events by the Escherichia coli Insertase YidC

Membrane protein translocation and insertion is a central issue in biology. Here we focus on a minimal system, the membrane insertase YidC of Escherichia coli that inserts small proteins into the cytoplasmic membrane. In a reconstituted system individual insertion processes were followed by single-pair fluorescence resonance energy transfer (FRET), with a pair of fluorophores on YidC and the substrate Pf3 coat protein. After addition of N-terminally labeled Pf3 coat protein a close contact to YidC at its cytoplasmic label was observed. This allowed to monitor the translocation of the N-terminal domain of Pf3 coat protein across the membrane in real time. Translocation occurred within milliseconds as the label on the N-terminal domain rapidly approached the fluorophore on the periplasmic domain of YidC at the trans side of the membrane. After the close contact, the two fluorophores separated, reflecting the release of the translocated Pf3 coat protein from YidC into the membrane bilayer. When the Pf3 coat protein was labeled C-terminally, no translocation of the label was observed although efficient binding to the cytoplasmic positions of YidC occurred.

Unbalanced Charge Distribution as a Determinant for Dependence of a Subset of Escherichia coli Membrane Proteins on the Membrane Insertase YidC

Unbalanced Charge Distribution as a Determinant for Dependence of a Subset of Escherichia coli Membrane Proteins on the Membrane Insertase YidC

Both YidC and SecYEG Are Required for Translocation of the Periplasmic Loops 1 and 2 of the Multispanning Membrane Protein TatC

TatC, a subunit of the twin arginine translocase, is a 6-membrane-spanning protein exposing three periplasmic loops. We have used TatC as a model system to examine how multispanning proteins insert into the membrane. To assay translocation of each of the three loops of TatC across the membrane, we used trypsin mapping, proteinase K mapping, and chemical modification methods. Here, we show that the signal recognition particle is required for targeting TatC to the inner membrane of Escherichiacoli. While translocation of loops 1 and 2 is strictly dependent on the Sec translocase and the YidC insertase, translocation of loop 3 does not depend on the translocase or insertase. None of the periplasmic loops require SecA or the proton motive force for membrane translocation. This work demonstrates a strategy where all the loops of a multispanning membrane protein can be monitored individually. The membrane translocation mechanism of each periplasmic loop can be complex with different energy and translocase requirements for a multispanning membrane protein.

The Membrane Insertase Oxa1 Is Required for Efficient Import of Carrier Proteins into Mitochondria

Oxa1 serves as a protein insertase of the mitochondrial inner membrane that is evolutionary related to the bacterial YidC insertase. Its activity is critical for membrane integration of mitochondrial translation products and conservatively sorted inner membrane proteins after their passage through the matrix. All Oxa1 substrates identified thus far have bacterial homologs and are of endosymbiotic origin. Here, we show that Oxa1 is critical for the biogenesis of members of the mitochondrial carrier proteins. Deletion mutants lacking Oxa1 show reduced steady-state levels and activities of the mitochondrial ATP/ADP carrier protein Aac2. To reduce the risk of indirect effects, we generated a novel temperature-sensitive oxa1 mutant that allows rapid depletion of a mutated Oxa1 variant in situ by mitochondrial proteolysis. Oxa1-depleted mitochondria isolated from this mutant still contain normal levels of the membrane potential and of respiratory chain complexes. Nevertheless, in vitro import experiments showed severely reduced import rates of Aac2 and other members of the carrier family, whereas the import of matrix proteins was unaffected. From this, we conclude that Oxa1 is directly or indirectly required for efficient biogenesis of carrier proteins. This was unexpected, since carrier proteins are inserted into the inner membrane from the intermembrane space side and lack bacterial homologs. Our observations suggest that the function of Oxa1 is relevant not only for the biogenesis of conserved mitochondrial components such as respiratory chain complexes or ABC transporters but also for mitochondria-specific membrane proteins of eukaryotic origin.

Dynamic Disulfide Scanning of the Membrane-inserting Pf3 Coat Protein Reveals Multiple YidC Substrate Contacts

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of ATP synthase and respiratory complexes is catalyzed by YidC. To investigate how YidC interacts with membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate Pf3 coat protein. The single cysteine mutants were expressed and analyzed for disulfide formation during 30 s of synthesis. The results show that the substrate contacts different YidC residues in four of the six transmembrane regions. The residues are located either in the region of the inner leaflet, in the center, as well as in the periplasmic leaflet, consistent with the hypothesis that YidC presents a hydrophobic platform for inserting membrane proteins. In a YidC mutant where most of the contacting residues were mutated to serines, YidC function was severely disturbed and no longer active in a complementation test, suggesting that the residues are important for function. In addition, a Pf3 mutant with a defect in membrane insertion was deficient to contact the periplasmic residues of YidC.

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by theEscherichia colisignal recognition particle

Protein insertion into the bacterial inner membrane is facilitated by SecYEG or YidC. Although SecYEG most likely constitutes the major integration site, small membrane proteins have been shown to integrate via YidC. We show that YidC can also integrate multispanning membrane proteins such as mannitol permease or TatC, which had been considered to be exclusively integrated by SecYEG. Only SecA-dependent multispanning membrane proteins strictly require SecYEG for integration, which suggests that SecA can only interact with the SecYEG translocon, but not with the YidC insertase. Targeting of multispanning membrane proteins to YidC is mediated by signal recognition particle (SRP), and we show by site-directed cross-linking that the C-terminus of YidC is in contact with SRP, the SRP receptor, and ribosomal proteins. These findings indicate that SRP recognizes membrane proteins independent of the downstream integration site and that many membrane proteins can probably use either SecYEG or YidC for integration. Because protein synthesis is much slower than protein transport, the use of YidC as an additional integration site for multispanning membrane proteins may prevent a situation in which the majority of SecYEG complexes are occupied by translating ribosomes during cotranslational insertion, impeding the translocation of secretory proteins.

Unbalanced Charge Distribution as a Determinant for Dependence of a Subset of Escherichia coli Membrane Proteins on the Membrane Insertase YidC

ABSTRACTMembrane proteins are involved in numerous essential cell processes, including transport, gene regulation, motility, and metabolism. To function properly, they must be inserted into the membrane and folded correctly. YidC, an essential protein in Escherichia coli with homologues in other bacteria, Archaea, mitochondria, and chloroplasts, functions by incompletely understood mechanisms in the insertion and folding of certain membrane proteins. Using a genome-scale approach, we identified 69 E. coli membrane proteins that, in the absence of YidC, exhibited aberrant localization by microscopy. Further examination of a subset revealed biochemical defects in membrane insertion in the absence of YidC, indicating their dependence on YidC for proper membrane insertion or folding. Membrane proteins possessing an unfavorable distribution of positively charged residues were significantly more likely to depend on YidC for membrane insertion. Correcting the charge distribution of a charge-unbalanced YidC-dependent membrane protein abrogated its requirement for YidC, while perturbing the charge distribution of a charge-balanced YidC-independent membrane protein rendered it YidC dependent, demonstrating that charge distribution can be a necessary and sufficient determinant of YidC dependence. These findings provide insights into a mechanism by which YidC promotes proper membrane protein biogenesis and suggest a critical function of YidC in all organisms and organelles that express it.

YidC-Driven Membrane Insertion of Single Fluorescent Pf3 Coat Proteins

The membrane insertion of single bacteriophage Pf3 coat proteins was observed by confocal fluorescence microscopy. Within seconds after addition of the purified and fluorescently labeled protein to liposomes or proteoliposomes containing the purified and reconstituted membrane insertase YidC of Escherichia coli, the translocation of the labeled residue was detected. The 50-amino-acid-long Pf3 coat protein was labeled with Atto520 and inserted into the proteoliposomes. Translocation of the dye into the proteoliposome was revealed by quenching the fluorescence outside of the vesicles. This allowed us to distinguish single Pf3 coat proteins that only bound to the surface of the liposomes from proteins that had inserted into the bilayer and translocated the dye into the lumen. The Pf3 coat protein required the presence of the YidC membrane insertase, whereas mutants that have a membrane-spanning region with an increased hydrophobicity were autonomously inserted into the liposomes without YidC.

Assembly of Bacterial Inner Membrane Proteins

Numerous membrane proteins form multisubunit protein complexes, which contain both integral and peripheral subunits, in addition to prosthetic groups. Bacterial membrane proteins are inserted into the inner membrane by the Sec translocase and YidC insertase. Their folding can be facilitated by YidC and the phospholipid phosphatidylethanolamine (PE). Glycine zippers and other motifs promote transmembrane-transmembrane (TM-TM) helix interactions that may lead to the formation of α-helical bundles of membrane proteins. During or after membrane insertion, the subunits of oligomeric membrane proteins must find each other to build the homo-oligomeric and the hetero-oligomeric membrane complexes. Although chaperones may function as assembly factors in the formation of the oligomer, many protein oligomers appear to fold and oligomerize spontaneously. Current studies show that most subunits of hetero-oligomers follow a sequential and ordered pathway to form the membrane protein complex. If the inserted protein is misfolded or the membrane protein is misassembled, quality control mechanisms exist that can degrade the proteins.

Substrate-Dependent Conformational Dynamics of the Escherichia coli Membrane Insertase YidC

The binding of Pf3 coat protein to the membrane insertase YidC from Escherichia coli induces a conformational change in the tertiary structure of the insertase, resulting in a quenching of the intrinsic tryptophan (Trp) fluorescence. Tryptophan mutants of YidC were generated to examine such conformational movements in detail with time-resolved and steady-state fluorescence spectroscopy. Ten of the 11 Trp residues within YidC were substituted to phenylalanines generating single Trp mutants either at position 354, 454, or 508. In addition, a double mutant with Trp residues at 332 and 334 was studied. Purified YidC mutants were reconstituted into DOPC/DOPG vesicles and titrated with a Trp-free mutant of Pf3 coat, enabling a detailed conformational study of the periplasmic P1, P2, and P3 domains of YidC before and after binding of substrate. Time-resolved fluorescence anisotropy revealed that the mobility of the residues W332/W334 and W508 was considerably increased after binding of Pf3 coat to the insertase. Furthermore, analysis of the fluorescence emission spectra and the decay times showed that all Trp residues are embedded in an equivalent environment that is a membrane/water interface.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

BackgroundFilamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles.ResultsThe amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags.ConclusionsOur results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

M13 Procoat Protein Insertion into YidC and SecYEG Proteoliposomes and Liposomes

M13 procoat protein was one of the first model proteins used to study bacterial membrane protein insertion. It contains a signal peptide of 23 amino acid residues and is not membrane targeted by the signal recognition particle. The translocation of its periplasmic domain is independent of the preprotein translocase (SecAYEG) but requires electrochemical membrane potential and the membrane insertase YidC of Escherichia coli. We show here that YidC is sufficient for efficient membrane insertion of the purified M13 procoat protein into energized YidC proteoliposomes. When no membrane potential is applied, the insertion is substantially reduced. Only in the presence of YidC, membrane insertion occurs if bilayer integrity is preserved and membrane potential is stable for more than 20 min. A mutant of the M13 procoat protein, H5EE, with two additional negatively charged residues in the periplasmic domain inserted into YidC proteoliposomes and SecYEG proteoliposomes with equal efficiencies. We conclude that the protein can use both the YidC-only pathway and the Sec pathway. This poses the questions of how procoat H5EE is inserted in vivo and how insertion pathways are selected in the cell.

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL: MscL does not need the insertase YidC for insertion in vitro

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.

Substrate-Induced Conformational Change of the Escherichia coli Membrane Insertase YidC

The membrane insertase YidC from Escherichia coli reversibly binds its substrate Pf3 coat protein. The effect of this initial binding process was examined in vitro by fluorescence quenching of the tryptophan (Trp) residues of YidC which are highly sensitive fluorescent probes for changes of the protein's tertiary structure. Membrane-reconstituted (in DOPC or DOPE/DOPG vesicles) as well as detergent-solubilized (C(12)PC) YidC was titrated with a Trp-free Pf3 coat mutant. Quenching of the intrinsic Trp fluorescence after titration indicates a change in the tertiary structure of YidC upon binding to the Pf3 coat substrate. Analysis of the binding curves taken from the fluorescence data yielded values for the dissociation constant (K(D)) in the range of 0.5-1.8 microM. Titration experiments with two Trp mutants reveal that the change in the tertiary structure involves mainly the membrane-spanning domain. The influence of the different environments on the secondary structure of YidC as well as of the YidC large periplasmic domain (P1) was investigated by circular dichroism (CD). The CD data show that the YidC secondary structure changes upon reconstitution into a membrane environment when compared to the detergent-solubilized state. In particular, the P1 domain of YidC is considerably affected by the detergent C(12)PC. This underlines the importance to study conformational changes with membrane-inserted proteins.

Subunit a of the F1F0 ATP Synthase Requires YidC and SecYEG for Membrane Insertion

The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F1F0 ATP synthase (F0a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F0a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F0a protein. These data demonstrate an extensive role of YidC in the assembly of the F0 sector of the F1F0 ATP synthase.

The Pf3 coat protein contacts TM1 and TM3 of YidC during membrane biogenesis

The coat protein of bacteriophage Pf3 is inserted into the plasma membrane of Escherichia coli by the insertase YidC. To identify which of the six transmembrane regions of YidC bind the single-spanning Pf3 coat protein during membrane protein biogenesis, we used the disulfide cross-linking approach. We generated single cysteines in each of the transmembrane regions of YidC and in the center of the hydrophobic region of Pf3 coat protein. We found that the substrate Pf3 coat contacts the first and third transmembrane segment (TM) of YidC as crosslinks between these two proteins can be formed in vivo during membrane biogenesis. A detailed disulfide-mapping study revealed that one face of TM3 of YidC makes contact with the Pf3 protein. Structured summary MINT- 6795850, MINT- 6795869, MINT- 6795912, MINT- 6795927, MINT- 6795942: Coat protein (uniprotkb: P03623) binds (MI: 0408) YidC (uniprotkb: P25714) by cross-linking studies (MI: 0030) MINT- 6795898: Coat protein (uniprotkb: P03623) binds (MI: 0408) Coat protein (uniprotkb: P03623) by cross-linking studies (MI: 0030)

Initial Binding Process of the Membrane Insertase YidC with Its Substrate Pf3 Coat Protein Is Reversible

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.