Signal Recognition Particle Pathway Research Articles

Examining SRP pathway function in mRNA localization to the endoplasmic reticulum.

Signal recognition particle (SRP) pathway function in protein translocation across the endoplasmic reticulum (ER) is well established; its role in RNA localization to the ER remains, however, unclear. In current models, mRNAs undergo translation- and SRP-dependent trafficking to the ER, with ER localization mediated via interactions between SRP-bound translating ribosomes and the ER-resident SRP receptor (SR), a heterodimeric complex comprising SRA, the SRP-binding subunit, and SRB, an integral membrane ER protein. To study SRP pathway function in RNA localization, SR knockout (KO) mammalian cell lines were generated and the consequences of SR KO on steady-state and dynamic mRNA localization examined. CRISPR/Cas9-mediated SRPRB KO resulted in profound destabilization of SRA. Pairing siRNA silencing of SRPRA in SRPRB KO cells yielded viable SR KO cells. Steady-state mRNA compositions and ER-localization patterns in parental and SR KO cells were determined by cell fractionation and deep sequencing. Notably, steady-state cytosol and ER mRNA compositions and partitioning patterns were largely unaltered by loss of SR expression. To examine SRP pathway function in RNA localization dynamics, the subcellular trafficking itineraries of newly exported mRNAs were determined by 4-thiouridine (4SU) pulse-labeling/4SU-seq/cell fractionation. Newly exported mRNAs were distinguished by high ER enrichment, with ER localization being SR-independent. Intriguingly, under conditions of translation initiation inhibition, the ER was the default localization site for all newly exported mRNAs. These data demonstrate that mRNA localization to the ER can be uncoupled from the SRP pathway function and reopen questions regarding the mechanism of RNA localization to the ER.

Chloroplast SRP43 and SRP54 independently promote thermostability and membrane binding of light-dependent protochlorophyllide oxidoreductases.

Protochlorophyllide oxidoreductase (POR), which converts protochlorophyllide into chlorophyllide, is the only light-dependent enzyme in chlorophyll biosynthesis. While its catalytic reaction and importance for chloroplast development are well understood, little is known about the post-translational control of PORs. Here, we show that cpSRP43 and cpSRP54, two components of the chloroplast signal recognition particle pathway, play distinct roles in optimizing the function of PORB, the predominant POR isoform in Arabidopsis. The chaperone cpSRP43 stabilizes the enzyme and provides appropriate amounts of PORB during leaf greening and heat shock, whereas cpSRP54 enhances its binding to the thylakoid membrane, thereby ensuring adequate levels of metabolic flux in late chlorophyll biosynthesis. Furthermore, cpSRP43 and the DnaJ-like protein CHAPERONE-LIKE PROTEIN of POR1 concurrently act to stabilize PORB. Overall, these findings enhance our understanding of the coordinating role of cpSPR43 and cpSRP54 in the post-translational control of chlorophyll synthesis and assembly of photosynthetic chlorophyll-binding proteins.

MRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By invivo and invitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

Metabolic Sensing of Extracytoplasmic Copper Availability via Translational Control by a Nascent Exported Protein.

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extracellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling, can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that the cotranslational export of the Rhodobacter capsulatus protein CutF regulates the translation of the downstream cutO-encoded multicopper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is cotranslationally exported by the signal recognition particle pathway. The binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its cotranslational export via its C-terminal ribosome stalling-like motif. This allows for the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO, which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and are often located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using the cotranslational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. IMPORTANCE Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation. Here, using Rhodobacter capsulatus, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a cotranslationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows for the unfolding of a downstream mRNA stem-loop and enables the translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for the integration of environmental signals into metabolic adaptations.

Synonymous Codons and Hydrophobicity Optimization of Post-translational Signal Peptide PelB Increase Phage Display Efficiency of DARPins.

DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelBWT and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelBWT, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.

Spatiotemporal kinetics of the SRP pathway in live E. coli cells

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of invitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining invivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Holin‐assisted bacterial recombinant protein export

A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage-encoded holin can be used to export proteins to the extracellular medium in both Gram-negative Escherichia coli and -positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic β-galatosidase (116 kDa), whose extracellular production reached 54.59 U/μL, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55-fold and fimbrial adhesin FaeG to 2.40-fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest. This article is protected by copyright. All rights reserved.

Localization of EccA3 at the growing pole in Mycobacterium smegmatis

BackgroundBacteria require specialized secretion systems for the export of molecules into the extracellular space to modify their environment and scavenge for nutrients. The ESX-3 secretion system is required by mycobacteria for iron homeostasis. The ESX-3 operon encodes for one cytoplasmic component (EccA3) and five membrane components (EccB3 – EccE3 and MycP3). In this study we sought to identify the sub-cellular location of EccA3 of the ESX-3 secretion system in mycobacteria.ResultsFluorescently tagged EccA3 localized to a single pole in the majority of Mycobacterium smegmatis cells and time-lapse fluorescent microscopy identified this pole as the growing pole. Deletion of ESX-3 did not prevent polar localization of fluorescently tagged EccA3, suggesting that EccA3 unipolar localization is independent of other ESX-3 components. Affinity purification - mass spectrometry was used to identify EccA3 associated proteins which may contribute to the localization of EccA3 at the growing pole. EccA3 co-purified with fatty acid metabolism proteins (FAS, FadA3, KasA and KasB), mycolic acid synthesis proteins (UmaA, CmaA1), cell division proteins (FtsE and FtsZ), and cell shape and cell cycle proteins (MurS, CwsA and Wag31). Secretion system related proteins Ffh, SecA1, EccA1, and EspI were also identified.ConclusionsTime-lapse microscopy demonstrated that EccA3 is located at the growing pole in M. smegmatis. The co-purification of EccA3 with proteins known to be required for polar growth, mycolic acid synthesis, the Sec secretion system (SecA1), and the signal recognition particle pathway (Ffh) also suggests that EccA3 is located at the site of active cell growth.

Deciphering the molecular organization of Get pathway chaperones through native top-down dissociation of multi-protein complexes

From regulating membrane trafficking to the release of neurotransmitters at the neuronal synapses, C-terminal tail-anchored membrane proteins (TAPs) play a central role in eukaryotic biology. Trafficking of TAPs to their target physiological membrane follows the usual anterograde trafficking route from the endoplasmic reticulum (ER), where they are first inserted. Nevertheless, unlike other membrane proteins, their insertion into the ER membrane is not mediated via the signal recognition particle (SRP) pathway.

A multifactorial assessment of the SRP pathway constituent FtsY as a vital mycobacterial constituent.

The signal recognition particle (SRP) system plays an imperative role in transporting the secretory protein to its intended location. The SRP pathway running in Mycobacterium tuberculosis constitutes FtsY (signal receptor), FfH (SRP), and 4.5S RNA in which signal receptor acts in the GTP-dependent manner. In this study, we are rendering the essential facts of FtsY with respect to mycobacterial growth. The growth study experiment showed that downexpressed FtsY slowed the growth of Mycobacterium smegmatis mc2 155 from the initial lag phase to stationary phase. Previously, we have showed that GTPase activity of FtsY is metal ion dependent and showed the maximum activity with 10mM magnesium. The effect of Mg2+ and Mn2+ on mycobacterial growth showed that Mg2+ did not affect the growth, whereas higher concentration of Mn2+ decreases the bacterial growth. After searching the inhibitor database, 14 GTPase and ATPase inhibitors, Mac0182344, ML141, ITX3, NAV_2729, Br-GTP, Rhosin_HCl, Mac0182099, CCG_50014, CID_1067700, Mac0174809, Nsc_23766, Berberine, Nexinhib20, and EHT1864, were found to interact with FtsY. Further, ML141 and NAV2729 found to decrease the enzymatic activity of FtsY as well as the mycobacterial growth. Therefore, the conclusive statement of the present study can be stated as that the FtsY plays major role in mycobacterial cell survival and ML141 and NAV2729 can be used to constrain the SRP pathway.

Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum.

Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.

Perturbation of the peptidoglycan network and utilization of the signal recognition particle-dependent pathway enhances the extracellular production of a truncational mutant of CelA in Escherichia coli.

Caldicellulosiruptor bescii is the most thermophilic, cellulolytic bacterium known and has the native ability to utilize unpretreated plant biomass. Cellulase A (CelA) is the most abundant enzyme in the exoproteome of C. bescii and is primarily responsible for its cellulolytic ability. CelA contains a family 9 glycoside hydrolase and a family 48 glycoside hydrolase connected by linker regions and three carbohydrate-binding domains. A truncated version of the enzyme (TM1) containing only the endoglucanase domain is thermostable and actively degrades crystalline cellulose. A catalytically active TM1 was successfully produced via the attachment of the PelB signal peptide (P-TM1), which mediates post-translational secretion via the SecB-dependent translocation pathway. We sought to enhance the extracellular secretion of TM1 using an alternative pathway, the signal recognition particle (SRP)-dependent translocation pathway. The co-translational extracellular secretion of TM1 via the SRP pathway (D-TM1) resulted in a specific activity that was 4.9 times higher than that associated with P-TM1 overexpression. In batch fermentations, the recombinant Escherichia coli overexpressing D-TM1 produced 1.86 ± 0.06 U/ml of TM1 in the culture medium, showing a specific activity of 1.25 ± 0.05 U/mg cell, 2.7- and 3.7-fold higher than the corresponding values of the strain overexpressing P-TM1. We suggest that the TM1 secretion system developed in this study can be applied to enhance the capacity of E. coli as a microbial cell factory for the extracellular secretion of this as well as a variety proteins important for commercial production.

Essential biochemical, biophysical and computational inputs on efficient functioning of Mycobacterium tuberculosis H37Rv FtsY

Mycobacterium tuberculosis (M. tuberculosis H37Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include Vmax and Km for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. kcat and catalytic efficiency of the reaction were 0.012±0.003 s−1 and 0.00047±0.0001 μM/s−1 respectively. These values were affected upon changing the standard conditions. Cations (Mg2+ and Mn2+) play important role in FtsY enzymatic activity as increasing Mg2+ decrease the activity. Mn2+on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence.

Signal Recognition Particle RNA Contributes to Oxidative Stress Response in Deinococcus radiodurans by Modulating Catalase Localization

The proper functioning of many proteins requires their transport to the correct cellular compartment or their secretion. Signal recognition particle (SRP) is a major protein transport pathway responsible for the co-translational movement of integral membrane proteins as well as periplasmic proteins. Deinococcus radiodurans is a ubiquitous bacterium that expresses a complex phenotype of extreme oxidative stress resistance, which depends on proteins involved in DNA repair, metabolism, gene regulation, and antioxidant defense. These proteins are located extracellularly or subcellularly, but the molecular mechanism of protein localization in D. radiodurans to manage oxidative stress response remains unexplored. In this study, we characterized the SRP complex in D. radiodurans R1 and showed that the knockdown (KD) of the SRP RNA (Qpr6) reduced bacterial survival under hydrogen peroxide and growth under chronic ionizing radiation. Through LC-mass spectrometry (MS/MS) analysis, we detected 162 proteins in the periplasm of wild-type D. radiodurans, of which the transport of 65 of these proteins to the periplasm was significantly reduced in the Qpr6 KD strain. Through Western blotting, we further demonstrated the localization of the catalases in D. radiodurans, DR_1998 (KatE1) and DR_A0259 (KatE2), in both the cytoplasm and periplasm, respectively, and showed that the accumulation of KatE1 and KatE2 in the periplasm was reduced in the SRP-defective strains. Collectively, this study establishes the importance of the SRP pathway in the survival and the transport of antioxidant proteins in D. radiodurans under oxidative stress.

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

The SRP54 GTPase is a key component of co-translational protein targeting by the signal recognition particle (SRP). Point mutations in SRP54 have been recently shown to lead to a form of severe congenital neutropenia displaying symptoms overlapping with those of Shwachman-Diamond syndrome. The phenotype includes severe neutropenia, exocrine pancreatic deficiency, and neurodevelopmental as well as skeletal disorders. Using a combination of X-ray crystallography, hydrogen-deuterium exchange coupled to mass spectrometry and complementary biochemical and biophysical methods, we reveal extensive structural defects in three disease-causing SRP54 variants resulting in critical protein destabilization. GTP binding is mostly abolished as a consequence of an altered GTPase core. The mutations located in conserved sequence fingerprints of SRP54 eliminate targeting complex formation with the SRP receptor as demonstrated in yeast and human cells. These specific defects critically influence the entire SRP pathway, thereby causing this life-threatening disease.

Live-cell Single-molecule Tracking for Protein Synthesis Kinetics Measurements

Our ability to relate results from biochemical experiments in the test tube, to the dynamic processes occurring in living cells is very limited. We have recently developed new experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. In a pioneering study, dye-labeled elongator tRNAPhe or initiator tRNAfMet were electroporated into E. coli cells, and tracked using super-resolved single-molecule microscopy. Using Hidden Markov Model based analysis of the diffusion trajectories, we estimated the dwell time of the tRNAs bound to ribosomes. Hence, with this approach we were able to directly measure kinetics of translation initiation and elongation at codon resolution inside living cells. In a follow-up study, the system was used to investigate the direct effect of the antibiotic chloramphenicol on ongoing protein synthesis. In this presentation, I will talk about our method, and in particular how we are now applying the method to study related biological phenomena. For example, by tracking key components of the SRP (Signal Recognition Particle) pathway, we measure the kinetics of co-translational translocon targeting of inner-membrane proteins.

A molecular recognition feature mediates ribosome-induced SRP-receptor assembly during protein targeting.

Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.

Loss of ALBINO3b Insertase Results in Truncated Light-Harvesting Antenna in Diatoms.

The family of chloroplast ALBINO3 (ALB3) proteins function in the insertion and assembly of thylakoid membrane protein complexes. Loss of ALB3b in the marine diatom Phaeodactylum tricornutum leads to a striking change of cell color from the normal brown to green. A 75% decrease of the main fucoxanthin-chlorophyll a/c-binding proteins was identified in the alb3b strains as the cause of changes in the spectral properties of the mutant cells. The alb3b lines exhibit a truncated light-harvesting antenna phenotype with reduced amounts of light-harvesting pigments and require a higher light intensity for saturation of photosynthesis. Accumulation of photoprotective pigments and light-harvesting complex stress-related proteins was not negatively affected in the mutant strains, but still the capacity for nonphotochemical quenching was lower compared with the wild type. In plants and green algae, ALB3 proteins interact with members of the chloroplast signal recognition particle pathway through a Lys-rich C-terminal domain. A novel conserved C-terminal domain was identified in diatoms and other stramenopiles, questioning if ALB3b proteins have the same interaction partners as their plant/green algae homologs.

Membrane proteomic analysis reveals overlapping and independent functions of Streptococcus mutans Ffh, YidC1, and YidC2.

A comparative proteomic analysis was utilized to evaluate similarities and differences in membrane samples derived from the cariogenic bacterium Streptococcus mutans, including the wild-type strain and four mutants devoid of protein translocation machinery components, specifically ∆ffh, ∆yidC1, ∆yidC2, or ∆ffh/yidC1. The purpose of this work was to determine the extent to which the encoded proteins operate individually or in concert with one another and to identify the potential substrates of the respective pathways. Ffh is the principal protein component of the signal recognition particle (SRP), while yidC1 and yidC2 are dual paralogs encoding members of the YidC/Oxa/Alb family of membrane-localized chaperone insertases. Our results suggest that the co-translational SRP pathway works in concert with either YidC1 or YidC2 specifically, or with no preference for paralog, in the insertion of most membrane-localized substrates. A few instances were identified in which the SRP pathway alone, or one of the YidCs alone, appeared to be most relevant. These data shed light on underlying reasons for differing phenotypic consequences of ffh, yidC1 or yidC2 deletion. Our data further suggest that many membrane proteins present in a ∆yidC2 background may be non-functional, that ∆yidC1 is better able to adapt physiologically to the loss of this paralog, that shared phenotypic properties of ∆ffh and ∆yidC2 mutants can stem from impacts on different proteins, and that independent binding to ribosomal proteins is not a primary functional activity of YidC2. Lastly, genomic mutations accumulate in a ∆yidC2 background coincident with phenotypic reversion, including an apparent W138R suppressor mutation within yidC1.

Dissecting structure/function relationship of Streptococcus mutans membrane protein chaperones/insertases, YidC1 and YidC2

BackgroundApproximately, one third of most bacterial open reading frames encode membrane proteins; therefore, understanding the machinery that translocates these proteins to their functional location is of great interest. Most membrane proteins are translocated co‐translationally by the signal recognition particle (SRP) pathway via the SecYEG translocon. Additionally the Oxa/Alb/YidC (mitochondria/chloroplast/bacteria) family of membrane‐localized protein chaperone/insertases assists in membrane protein translocation in a Sec‐dependent manner, or independently if the proteins are small with two or fewer transmembrane (TM) domains. Most gram‐negative bacteria contain a single yidC whose deletion is lethal. Most gram‐positive bacteria contain two or more YidCs. The cariogenic bacterium S. mutans encodes YidC1 and YidC2, each with similar 5 TM domain topologies. Deletion of yidC2 results in a severe phenotype similar to disruption of the SRP pathway including impaired growth, sensitivity to acid, osmotic, and oxidative stresses, and loss of competence and mutacin production. Deletion of DyidC1 leads to increased localization of surface proteins and hyperadherence. The goal of this study is to better understand the structure and function of YidC1 and YidC2 as related to their known phenotypes.MethodsThe significance of the two cytoplasmic loops, C1 and C2, of YidC1 and YidC2 were evaluated in domain swapping experiments in which DNA segments encoding one or both C1 and C2 domains of yidC1 were incorporated into yidC2 and vice versa. Chimeric genes were integrated into the non‐essential gtfA locus of the DyidC1 and DyidC2 strains. Next the residual endogenous yidC was deleted, thus generating strains with a sole chimeric YidC for phenotypic analyses. In addition, a naturally‐acquired W138R point mutation within yidC1 was identified in a phenotypically‐reverted DyidC2 mutant; therefore, the significance of this residue was evaluated as well.Results and ConclusionsAn S. mutans strain producing a chimeric YidC2 harboring the C1 loop of YidC1 was similar to the DyidC2 strain with respect to sensitivity to acid and osmotic stress suggesting a functional contribution of YidC2's C1 loop to stress tolerance. Transfer of YidC2 C1 into YidC1 enabled YidC1 to rescue acid and osmotic stress sensitivity. Also, a deliberately engineered W138R point mutation within YidC1 enabled it to rescue growth and stress phenotypes of the DyidC2 mutant. Sequence comparison revealed that the YidC1 tryptophan 138 residue corresponds to serine 152 in YidC2, the only polar residue within its TM3. Additionally, engineered W138K/S mutations within YidC1 also enabled rescue of DyidC2‐associated phenotypes. These data reveal that functional activities unique to YidC2 reside within both its C1 domain and TM3 that can be transferred to YidC1.Support or Funding InformationNIH grant: R01 DE08007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.