Signal Recognition Particle Pathway Research Articles

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.

Interactions of protein translocation machinery components of Streptococcus mutans

BackgroundPivotal virulence properties of the cariogenic bacterium Streptococcus mutans depend on secreted and integral membrane proteins. Bacterial protein trafficking involves the signal recognition particle (SRP) pathway components Ffh, scRNA and FtsY, the SecYEG translocon, the SecA motor protein, and membrane‐localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The phenotype of a DyidC2 mutant largely parallels that of Dffh, while that of DyidC1 is far less severe. Also in S. mutans, YlxM acts as an accessory factor of the SRP that modulates GTP hydrolysis activities of Ffh and FtsY. The goal of this study is to identify interactions among S. mutans transport machinery components to better understand the respective functions of YidC1 and YidC2 alone and in concert with the SRP, ribosome, and/or Sec translocon that can affect the destiny of translocated proteins.MethodsA chemical cross‐linking approach was employed, whereby whole cell lysates (wcl) were treated with formaldehyde followed by Western blotting using anti‐Ffh, FtsY, YidC1 and YidC2 antibodies and mass spectrometry (MS) analysis of corresponding gel‐shifted bands. Wcl from WT and DyidC2 strains were also reacted with a‐YidC2 antibodies coupled to magnetic Dynabeads™ and co‐captured proteins identified by MS. In addition, biolayer interferometry (BLI) was used to evaluate specific interactions of the C‐terminal tails of YidC1 and YidC2, engineered as glutathione‐S‐transferase fusion proteins (GST‐C1 and GST‐C2), with the SRP components Ffh and YlxM. The same GST‐constructs were also subjected to 2D Difference Gel Electrophoresis (DIGE), in which each was reacted with wcl, captured with glutathione affinity resin, and labelled individually with fluorescent dyes. Spots unique to the GST‐C1 and/or GST‐C2 samples were analysed by MS.Results and ConclusionBand‐shift patterns detected with anti‐Ffh, FtsY and YidC2 antisera were similar to one another and distinct from that of anti‐YidC1. Cross‐linked proteins included elongation factor Tu, DNA‐directed RNA polymerase subunit b, and HSP 70. Immunocaptured proteins from anti‐YidC2 Dynabeads™ included two small, transmembrane proteins, LemA and SMU_1719c, which may represent translocation substrates. Both GST‐C1 and GST‐C2 interacted with Ffh by BLI with binding influenced by YlxM. Over one hundred spots were identified by DIGE. Among differentially identified proteins in the GST‐C1 sample were DnaK, RNA polymerase subunit d, and cell division protein FtsH. Ribosomal proteins S7 and L13 were prevalent in spots of the GST‐C2 sample, whereas ribosomal protein L2, DNA polymerase III g/t subunit, and the Cas5 homolog were all more abundant in spots from GST‐C1 and GST‐C2 samples compared to GST. This information suggests a coupled cellular machinery that integrates transcription and translation with protein transport and folding.Support or Funding InformationNIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

IN SILICO STRUCTURAL CHARACTERIZATION OF L. lactis subsp. cremoris MG1363 FFH-FTSY COMPLEX IN PROTEIN TARGETING INTERACTION

In bacteria, gene conservation and experimental data show that Lactococcus lactis has the simplest version of protein secretion system compared to Escherichia coli and Bacillus subtilis whose systems are more complex. L. lactis only possess the signal recognition particle (SRP) pathway, where the specific interaction of Ffh and FtsY is known to be essential for the efficiency and fidelity of its protein targeting. Therefore, modelling and structural characterization study of Ffh and FtsY will give an idea of its crucial region and amino acids that are critical in Ffh-FtsY interaction during protein targeting. This work is the first attempt to model L. lactis Ffh-FtsY complex, which was derived by computational docking, where a blind dock was applied. Results showed that the complex interface was predominantly stabilized by four hydrophobic interactions and 17 hydrogen bonds, where these putative binding interfaces are mostly confined at the motifs II and III in each G domain of Ffh and FtsY. Several residues were expected to play important roles in initiating or regulating guanosine triphosphate hydrolysis, including residue R142. This structural information will allow for the rational design of L. lactis Ffh-FtsY association in the future.

The Escherichia coli SRP Receptor Forms a Homodimer at the Membrane

The Escherichia coli signal recognition particle (SRP) receptor, FtsY, plays a fundamental role in co-translational targeting of membrane proteins via the SRP pathway. Efficient targeting relies on membrane interaction of FtsY and heterodimerization with the SRP protein Ffh, which is driven by detachment of α helix (αN1) in FtsY. Here we show that apart from the heterodimer, FtsY forms a nucleotide-dependent homodimer on the membrane, and upon αN1 removal also in solution. Homodimerization triggers reciprocal stimulation of GTP hydrolysis and occurs invivo. Biochemical characterization together with integrative modeling suggests that the homodimer employs the same interface as the heterodimer. Structure determination of FtsY NG+1 with GMPPNP shows that a dimerization-induced conformational switch of the γ-phosphate is conserved in Escherichia coli, filling an important gap in SRP GTPase activation. Our findings add to the current understanding of SRP GTPases and may challenge previous studies that did not consider homodimerization of FtsY.

Cotranslational protein targeting to the membrane: Nascent-chain transfer in a quaternary complex formed at the translocon

Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG–FtsY binding to RNC–SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the translocon and repositioning of SRP in the post-transfer complex.

Chloroplast SRP43 acts as a chaperone for glutamyl-tRNA reductase, the rate-limiting enzyme in tetrapyrrole biosynthesis

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

Characterization of FtsY, its interaction with Ffh, and proteomic identification of their potential substrates in Mycobacterium tuberculosis.

The universally conserved signal recognition particle (SRP) pathway that mediates co-translational targeting of membrane and secretory proteins is essential for eukaryotic and prokaryotic cells. The Mycobacterium tuberculosis SRP pathway consists of 2 proteins, Ffh and FtsY, and a 4.5S RNA molecule. Although the Escherichia coli SRP pathway is well studied, understanding of the M. tuberculosis SRP pathway components is very limited. In this study, we have overexpressed and characterized the M. tuberculosis SRP receptor (SR) FtsY as a GTP binding protein. Further, we established the direct protein-protein interaction between Ffh and FtsY. The Ffh-FtsY complex formation resulted in mutual stimulation of their GTP hydrolysis activity. We also attempted to biochemically characterize the SRP components by constructing the antisense gene knockdown strains of ffh and ftsY in M. tuberculosis. Loss of ffh and ftsY resulted in a decreased in vitro growth rate of the antisense ffh strain as compared with the antisense ftsY strain. Finally, 2-D gel electrophoresis of antisense depleted ffh and ftsY strains identified differential expression of 14 proteins.

Deletion of the chloroplast LTD protein impedes LHCI import and PSI-LHCI assembly in Chlamydomonas reinhardtii.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane

BackgroundIn thylakoid membrane, each monomer of the dimeric complex of cytochrome b6f is comprised of eight subunits that are both nucleus- and plastid-encoded. Proper cytochrome b6f complex integration into the thylakoid membrane requires numerous regulatory factors for coordinated transport, insertion and assembly of the subunits. Although, the chloroplast-encoded cytochrome b6f subunit IV (PetD) consists of three transmembrane helices, the signal and the mechanism of protein integration into the thylakoid membrane have not been identified.ResultsHere, we demonstrate that the native PetD subunit cannot incorporate into the thylakoid membranes spontaneously, but that proper integration occurs through the post-translational signal recognition particle (SRP) pathway. Furthermore, we show that PetD insertion into thylakoid membrane involves the coordinated action of cpFTSY, cpSRP54 and ALB3 insertase.ConclusionsPetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex. This data provides a new insight into the molecular mechanisms by which membrane proteins integration into the thylakoid membrane is accomplished and is not limited to PetD.

Sorting of SEC translocase SCY components to different membranes in chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately routed in order to establish and maintain the highly differentiated membranes characteristic of these organelles. Little is known about the targeting information or pathways involved, especially in the case of proteins with multiple transmembrane domains. We have studied targeting of the SCY components of the two SEC translocases in chloroplasts. SCY1 and SCY2 share a similar, highly conserved structure with 10 transmembrane domains, but are targeted to different membranes: the thylakoids and inner envelope, respectively. We used protoplast transfections and a confocal microscopy imaging assay in combination with a domain-swapping approach to investigate sorting pathways and identify important targeting elements in these proteins. We show that the N-terminal region of SCY1 contains targeting determinants that allow SCY1 to be recruited to the signal-recognition particle pathway. In addition, substituting the N-terminal region of SCY1 for the N-terminal region of SCY2 causes SCY2 to be displaced out of the inner envelope. The region of SCY2 that contains transmembrane domains 3 and 4 is necessary for localization to the inner envelope and may serve as a membrane anchor, enhancing the integration of other transmembrane domains via either stop-transfer or post-import mechanisms.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

Cell biology: Sort of unexpected.

To reach the cell surface, membrane proteins are first targeted to an organelle called the endoplasmic reticulum. Several targeting pathways are known, but it now emerges that there is yet another pathway. See Letter p.134 On their way to various membranes within cells, cellular proteins are targeted to the endoplasmic reticulum, where they undergo maturation steps. Such endoplasmic reticulum targeting can occur either simultaneously with the translation of the proteins, mediated by the signal recognition particle (SRP) pathway, or following translation through the TRC40/GET pathway. However, many proteins do not depend on either of these pathways, indicating the existence of alternative modes of protein transport into the endoplasmic reticulum. Using a systematic screen in yeast, Maya Schuldiner and colleagues identify three proteins that function together—and in parallel with SRP and GET—to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which they call SND (SRP-iNDependent targeting) proteins, can compensate for the loss of both the SRP and GET pathways, acting as a backup targeting system. These data point to the robustness of the endoplasmic reticulum targeting apparatus.

Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction.

Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway.

The Chloroplast SRP Systems of Chaetosphaeridium globosum and Physcomitrella patens as Intermediates in the Evolution of SRP-Dependent Protein Transport in Higher Plants.

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., Chlamydomonas reinhardtii). In this study, we biochemically characterized the cpSRP system of the charophyte Chaetosphaeridium globosum and the bryophyte Physcomitrella patens. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 (Kd ~10 μM) in Chaetosphaeridium globosum and Physcomitrella patens as well as relatively low affinity binding of cpSRP54 to cpSRP RNA (Kd ~1 μM) in Physcomitrella patens. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the Physcomitrella patens cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA.

Cotranslational Intersection between the SRP and GET Targeting Pathways to the Endoplasmic Reticulum of Saccharomyces cerevisiae.

Targeting of transmembrane proteins to the endoplasmic reticulum (ER) proceeds via either the signal recognition particle (SRP) or the guided entry of tail-anchored proteins (GET) pathway, consisting of Get1 to -5 and Sgt2. While SRP cotranslationally targets membrane proteins containing one or multiple transmembrane domains, the GET pathway posttranslationally targets proteins containing a single C-terminal transmembrane domain termed the tail anchor. Here, we dissect the roles of the SRP and GET pathways in the sorting of homologous, two-membrane-spanning K(+) channel proteins termed Kcv, Kesv, and Kesv-VV. We show that Kcv is targeted to the ER cotranslationally via its N-terminal transmembrane domain, while Kesv-VV is targeted posttranslationally via its C-terminal transmembrane domain, which recruits Get4-5/Sgt2 and Get3. Unexpectedly, nascent Kcv recruited not only SRP but also the Get4-5 module of the GET pathway to ribosomes. Ribosome binding of Get4-5 was independent of Sgt2 and was strongly outcompeted by SRP. The combined data indicate a previously unrecognized cotranslational interplay between the SRP and GET pathways.

The bacterial SRP receptor, FtsY, is activated on binding to the translocon.

Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid-embedded translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the translocon. We propose that FtsY activation at the translocon ensures that ribosome-SRP complexes are directed to available translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the translocon achieved.

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY.

Integral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

AbstractIntegral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)-dependent secretion pathway by deletion of rrsE in Escherichia coli.

Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

YGL9, encoding the putative chloroplast signal recognition particle 43 kDa protein in rice, is involved in chloroplast development

The nuclear-encoded light-harvesting chlorophyll a/b-binding proteins (LHCPs) are specifically translocated from the stroma into the thylakoid membrane through the chloroplast signal recognition particle (cpSRP) pathway. The cpSRP is composed of a cpSRP43 protein and a cpSRP54 protein, and it forms a soluble transit complex with LHCP in the chloroplast stroma. Here, we identified the YGL9 gene that is predicted to encode the probable rice cpSRP43 protein from a rice yellow-green leaf mutant. A phylogenetic tree showed that an important conserved protein family, cpSRP43, is present in almost all green photosynthetic organisms such as higher plants and green algae. Sequence analysis showed that YGL9 comprises a chloroplast transit peptide, three chromodomains and four ankyrin repeats, and the chromodomains and ankyrin repeats are probably involved in protein-protein interactions. Subcellular localization showed that YGL9 is localized in the chloroplast. Expression pattern analysis indicated that YGL9 is mainly expressed in green leaf sheaths and leaves. Quantitative real-time PCR analysis showed that the expression levels of genes associated with pigment metabolism, chloroplast development and photosynthesis were distinctly affected in the ygl9 mutant. These results indicated that YGL9 is possibly involved in pigment metabolism, chloroplast development and photosynthesis in rice.