Identification of Bacillus subtilis YidC Substrates Using a MifM-instructed Translation Arrest-based Reporter

YidC is a member of the YidC/Oxa1/Alb3 protein family that is crucial for membrane protein biogenesis in the bacterial plasma membrane. While YidC facilitates the folding and complex assembly of membrane proteins along with the Sec translocon, it also functions as a Sec-independent membrane protein insertase in the YidC-only pathway. However, little is known about how membrane proteins are recognized and sorted by these pathways, especially in Gram-positive bacteria, for which only a small number of YidC substrates have been identified to date. In this study, we aimed to identify Bacillus subtilis membrane proteins whose membrane insertion depends on SpoIIIJ, the primary YidC homolog in B. subtilis. We took advantage of the translation arrest sequence of MifM, which can monitor YidC-dependent membrane insertion. Our systematic screening identified eight membrane proteins as candidate SpoIIIJ substrates. Results of our genetic study also suggest that the conserved arginine in the hydrophilic groove of SpoIIIJ is crucial for the membrane insertion of the substrates identified here. However, in contrast to MifM, a previously identified YidC substrate, the importance of the negatively charged residue on the substrates for membrane insertion varied depending on the substrate. These results suggest that B. subtilis YidC uses substrate-specific interactions to facilitate membrane insertion.     

A hydrophilic microenvironment in the substrate-translocating groove of the YidC membrane insertase is essential for enzyme function

The YidC family of proteins are membrane insertases that catalyze the translocation of the periplasmic domain of membrane proteins via a hydrophilic groove located within the inner leaflet of the membrane. All homologs have a strictly conserved, positively charged residue in the center of this groove. In Bacillus subtilis, the positively charged residue has been proposed to be essential for interacting with negatively charged residues of the substrate, supporting a hypothesis that YidC catalyzes insertion via an early-step electrostatic attraction mechanism. Here, we provide data suggesting that the positively charged residue is important not for its charge but for increasing the hydrophilicity of the groove. We found that the positively charged residue is dispensable for Escherichia coli YidC function when an adjacent residue at position 517 was hydrophilic or aromatic, but was essential when the adjacent residue was apolar. Additionally, solvent accessibility studies support the idea that the conserved positively charged residue functions to keep the top and middle of the groove sufficiently hydrated. Moreover, we demonstrate that both the E. coli and Streptococcus mutans YidC homologs are functional when the strictly conserved arginine is replaced with a negatively charged residue, provided proper stabilization from neighboring residues. These combined results show that the positively charged residue functions to maintain a hydrophilic microenvironment in the groove necessary for the insertase activity, rather than to form electrostatic interactions with the substrates.     

The charge distribution in the cytoplasmic loop of subunit C of the F1F0 ATPase is a determinant for YidC targeting

YidC is a member of the Oxa1 family of proteins that facilitates the membrane insertion of a subset of inner membrane proteins in Escherichia coli. YidC acts as an insertase for membrane insertion of subunit c of the F(1)F(0) ATP synthase (F(0)c), but the requirements for substrate recognition have remained unclear. Here, we have analyzed the role of the charged aminoacyl residues in F(0)c in YidC targeting and membrane insertion. Binding experiments demonstrate that F(0)c is targeted directly to YidC without the presence of a stable lipid surface-bound intermediate. Positive charges in the cytoplasmic loop of F(0)c are important determinants for YidC binding and subsequent membrane insertion. These data support a model in which F(0)c binds directly to YidC prior to its membrane insertion.     

Isolation of cold-sensitive yidC mutants provides insights into the substrate profile of the YidC insertase and the importance of transmembrane 3 in YidC function

YidC, a 60-kDa integral membrane protein, plays an important role in membrane protein insertion in bacteria. YidC can function together with the SecYEG machinery or operate independently as a membrane protein insertase. In this paper, we describe two new yidC mutants that lead to a cold-sensitive phenotype in bacterial cell growth. Both alleles impart a cold-sensitive phenotype and result from point mutations localized to the third transmembrane (TM3) segment of YidC, indicating that this region is crucial for YidC function. We found that the yidC(C423R) mutant confers a weak phenotype on membrane protein insertion while a yidC(P431L) mutant leads to a stronger phenotype. In both cases, the affected substrates include the Pf3 coat protein and ATP synthase F₁F_o subunit c (F_oC), while CyoA (the quinol binding subunit of the cytochrome bo3 quinol oxidase complex) and wild-type procoat are slightly affected or not affected in either cold-sensitive mutant. To determine if the different substrates require various levels of YidC activity for membrane insertion, we performed studies where YidC was depleted using an arabinose-dependent expression system. We found that −3M-PC-Lep (a construct with three negatively charged residues inserted into the middle of the procoat-Lep [PC-Lep] protein) and Pf3 P2 (a construct with the Lep P2 domain added at the C terminus of Pf3 coat) required the highest amount of YidC and that CyoA-N-P2 (a construct with the amino-terminal part of CyoA fused to the Lep P2 soluble domain) and PC-Lep required the least, while F_oC required moderate YidC levels. Although the cold-sensitive mutations can preferentially affect one substrate over another, our results indicate that different substrates require different levels of YidC activity for membrane insertion. Finally, we obtained several intragenic suppressors that overcame the cold sensitivity of the C423R mutation. One pair of mutations suggests an interaction between TM2 and TM3 of YidC. The studies reveal the critical regions of the YidC protein and provide insight into the substrate profile of the YidC insertase.     

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.     

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.     

Membrane protein insertase YidC in bacteria and archaea

The insertion of proteins into the prokaryotic plasma membrane is catalyzed by translocases and insertases. On one hand, the Sec translocase operates as a transmembrane channel that can open laterally to first bind and then release the hydrophobic segments of a substrate protein into the lipid bilayer. On the other hand, YidC insertases interact with their substrates in a groove‐like structure at an amphiphilic protein–lipid interface thus allowing the transmembrane segments of the substrate to slide into the lipid bilayer. The recently published high‐resolution structures of YidC provide new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane.     

Mechanisms of YidC-mediated insertion and assembly of multimeric membrane protein complexes

The YidC protein fulfills a dual and essential role in the assembly of inner membrane proteins in Escherichia coli. Besides interacting with transmembrane segments of newly synthesized membrane proteins that insert into the membrane via the SecYEG complex, YidC also functions as an independent membrane protein insertase and assists in membrane protein folding. Here, we discuss the mechanisms of YidC substrate recognition and membrane insertion with emphasis on its role in the assembly of multimeric membrane protein complexes such as the F1F0-ATP synthase.     

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.     

Different regions of the nonconserved large periplasmic domain of Escherichia coli YidC are involved in the SecF interaction and membrane insertase activity

The YidC protein of Escherichia coli is required for inserting Sec-independent membrane proteins and has a supportive role for the insertion of Sec-dependent proteins into the membrane bilayer. Because a portion of YidC copurifies with the Sec translocase, this interaction might be necessary to assist in the membrane insertion of Sec-dependent proteins. This study describes a deletion analysis that investigates which parts of YidC are required for its interaction with the SecDF complex of the Sec translocase and for the function of YidC as an insertase for the Sec-dependent membrane proteins. The results suggest that the first periplasmic region, which includes residues 24-346, is required for the interaction of YidC with the Sec translocase, in particular with the SecF protein. Further studies showed that residues 215-265 of YidC are sufficient for SecF binding. Surprisingly, the interaction of YidC with SecF is not critical for cell viability as YidC, lacking residues 24-264, was fully functional to support the growth of E. coli. It was also observed that this YidC mutant was fully functional to insert the Sec-dependent subunit A of the F(1)F(o) ATP synthase and an M13 procoat derivative, as well as the Sec-independent M13 procoat protein and subunit C of the ATP synthase. Only when additional residues of the periplasmic region were deleted (265-346) was the membrane insertase function of YidC inhibited.     

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.     

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.     

Promiscuous targeting of polytopic membrane proteins to SecYEG or YidC by the Escherichia coli signal 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.     

Post-translational membrane insertion of an endogenous YidC substrate

Membrane protein insertion is controlled by proteinaceous factors embedded in the lipid bilayer. Bacterial inner membrane proteins utilise the Sec translocon as the major facilitator of insertion; however some proteins are Sec independent and instead require only YidC. A common feature of YidC substrates is the exposure of a signal anchor sequence when translation is close to completion; this allows minimal time for targeting and favours a post-translational insertion mechanism. Despite this there is little evidence of YidC's post-translational activity. Here we develop an experimental system that uncouples translation and insertion of the endogenous YidC substrate F₀c (subunit c of the F₀F₁ ATP synthase). In this process we (i) develop a novel one step purification method for YidC, including an on column membrane reconstitution, (ii) isolate a soluble form of F₀c and (iii) show that incubation of F₀c with YidC proteoliposomes results in a high level of membrane integration. Conformational analyses of inserted F₀c through Blue Native PAGE and fluorescence quenching reveal a native, oligomerised structure. These data show that YidC can act as a post-translational insertase, a finding which could explain the absence of a ribosome binding domain on YidC. This correlates with the post-translational activity of other YidC family members lacking the ribosome binding domain.     

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.     

Biogenesis of YidC Cytoplasmic Membrane Substrates Is Required for Positioning of Autotransporter IcsA at Future Poles

Localization of proteins to specific sites within bacterial cells is often critical to their function. In rod-shaped bacteria, proteins involved in diverse and important cell processes localize to the cell poles. The molecular mechanisms by which these proteins are targeted to the pole, however, are poorly understood. The Shigella autotransporter protein IcsA, which is localized to the pole on the surface of the bacterium, is targeted to the pole in the cytoplasm by a mechanism that is conserved across multiple Gram-negative bacterial species and has thus served as an important and informative model for studying polar localization. We present evidence that in Escherichia coli, the establishment of polar positional information recognized by IcsA requires the activity of the cytoplasmic membrane protein insertase YidC. We show that the role of YidC in IcsA localization is independent of the cell septation and cytokinesis proteins FtsQ and FtsEX. FtsQ is required for polar localization of IcsA and, based on cross-linking studies, is inserted in the vicinity of YidC, but, we find, is not dependent on YidC for membrane insertion. FtsEX is a YidC substrate, but we find that it is not required for polar localization of IcsA. These findings indicate that polar positional information recognized by IcsA depends on one or more membrane proteins that require YidC for proper membrane insertion.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

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.     

MifM Monitors Total YidC Activities of Bacillus subtilis,Including That of YidC2, the Target of Regulation

The YidC/Oxa1/Alb3 family proteins are involved in membrane protein biogenesis in bacteria, mitochondria, and chloroplasts. Recent studies show that YidC uses a channel-independent mechanism to insert a class of membrane proteins into the membrane. Bacillus subtilis has two YidC homologs, SpoIIIJ (YidC1) and YidC2 (YqjG); the former is expressed constitutively, while the latter is induced when the SpoIIIJ activity is compromised. MifM is a substrate of SpoIIIJ, and its failure in membrane insertion is accompanied by stable ribosome stalling on the mifM-yidC2 mRNA, which ultimately facilitates yidC2 translation. While mutational inactivation of SpoIIIJ has been known to induce yidC2 expression, here, we show that the level of this induction is lower than that observed when the membrane insertion signal of MifM is defective. Moreover, this partial induction of YidC2 translation is lowered further when YidC2 is overexpressed in trans. These results suggest that YidC2 is able to insert MifM into the membrane and to release its translation arrest. Thus, under SpoIIIJ-deficient conditions, YidC2 expression is subject to MifM-mediated autogenous feedback repression. Our results show that YidC2 uses a mechanism that is virtually identical to that used by SpoIIIJ; Arg75 of YidC2 in its intramembrane yet hydrophilic cavity is functionally indispensable and requires negatively charged residues of MifM as an insertion substrate. From these results, we conclude that MifM monitors the total activities of the SpoIIIJ and the YidC2 pathways to control the synthesis of YidC2 and to maintain the cellular capability of the YidC mode of membrane protein biogenesis.     

YidC family members are involved in the membrane insertion, lateral integration, folding, and assembly of membrane proteins

Members of the YidC family exist in all three domains of life, where they control the assembly of a large variety of membrane protein complexes that function as transporters, energy devices, or sensor proteins. Recent studies in bacteria have shown that YidC functions on its own as a membrane protein insertase independent of the Sec protein-conducting channel. YidC can also assist in the lateral integration and folding of membrane proteins that insert into the membrane via the Sec pathway.