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Inner membrane YfgM–PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation

2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês

10.1016/j.jbc.2022.102572

ISSN

1083-351X

Autores

Ryoji Miyazaki, Mengting Ai, Natsuko Tanaka, Takehiro Suzuki, Naoshi Dhomae, Tomoya Tsukazaki, Yoshinori Akiyama, Hiroyuki Mori,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD–YfgM complex formation as well as the molecular mechanisms of PpiD–YfgM and PpiD/YfgM–Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD–YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon. PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD–YfgM complex formation as well as the molecular mechanisms of PpiD–YfgM and PpiD/YfgM–Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of Vibrio protein export monitoring polypeptide (VemP), a Vibrio secretory protein, in both E. coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photocrosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD–YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating their proper interactions with the Sec translocon. In gram-negative bacteria, around 30% of proteins are localized and function at the cell surface on the inner membrane (IM), periplasm, or outer membrane. After being synthesized in the cytoplasm, these proteins must be translocated across and/or integrated within the IM to reach their destination. The evolutionally conserved SecY/E/G (Sec) translocon, which consists of three integral IM proteins (SecY, SecE, and SecG), creates a narrow channel for the membrane translocation of newly synthesized polypeptides (1Van den Berg B. Clemons W.M.J. Collinson I. Modis Y. Hartmann E. Harrison S.C. et al.X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Crossref PubMed Scopus (1004) Google Scholar, 2Mori H. Ito K. The Sec protein-translocation pathway.Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 3Rapoport T.A. Li L. Park E. Structural and mechanistic insights into protein translocation.Annu. Rev. Cell Dev. Biol. 2017; 33: 369-390Crossref PubMed Scopus (193) Google Scholar, 4Tanaka Y. Sugano Y. Takemoto M. Mori T. Furukawa A. Kusakizako T. et al.Crystal structures of SecYEG in lipidic cubic phase elucidate a precise resting and a peptide-bound state.Cell Rep. 2015; 13: 1561-1568Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). SecY, the central component of the translocon, contains 10 transmembrane (TM) segments that form the protein translocation channel (2Mori H. Ito K. The Sec protein-translocation pathway.Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 5Akiyama Y. Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli.EMBO J. 1987; 6: 3465-3470Crossref PubMed Scopus (173) Google Scholar), whereas SecE and SecG peripherally associate with SecY (2Mori H. Ito K. The Sec protein-translocation pathway.Trends Microbiol. 2001; 9: 494-500Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 4Tanaka Y. Sugano Y. Takemoto M. Mori T. Furukawa A. Kusakizako T. et al.Crystal structures of SecYEG in lipidic cubic phase elucidate a precise resting and a peptide-bound state.Cell Rep. 2015; 13: 1561-1568Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Since the Sec translocon is a passive channel, an essential ATPase motor (SecA) (6Lill R. Cunningham K. Brundage L.A. Ito K. Oliver D. Wickner W. SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.EMBO J. 1989; 8: 961-966Crossref PubMed Scopus (355) Google Scholar, 7Economou A. Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion.Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 8Zimmer J. Nam Y. Rapoport T.A. Structure of a complex of the ATPase SecA and the protein-translocation channel.Nature. 2008; 455: 936-943Crossref PubMed Scopus (359) Google Scholar) and an IM-integrated proton motive force–driven motor (SecD/F complex) (9Tsukazaki T. Structure-based working model of SecDF, a proton-driven bacterial protein translocation factor.FEMS Microbiol. Lett. 2018; 365: fny112Crossref PubMed Scopus (33) Google Scholar, 10Tsukazaki T. Mori H. Echizen Y. Ishitani R. Fukai S. Tanaka T. et al.Structure and function of a membrane component SecDF that enhances protein export.Nature. 2011; 474: 235-238Crossref PubMed Scopus (177) Google Scholar) drive polypeptide movement through the Sec translocon from the cytoplasmic and periplasmic sides, respectively. In addition, the Sec translocon can interact with the membrane protein insertase, YidC, to integrate membrane proteins into the IM (11Samuelson J.C. Chen M. Jiang F. Möller I. Wiedmann M. Kuhn A. et al.YidC mediates membrane protein insertion in bacteria.Nature. 2000; 406: 637-641Crossref PubMed Scopus (432) Google Scholar, 12Kumazaki K. Chiba S. Takemoto M. Furukawa A. Nishiyama K. Sugano Y. et al.Structural basis of Sec-independent membrane protein insertion by YidC.Nature. 2014; 509: 516-520Crossref PubMed Scopus (167) Google Scholar, 13Kumazaki K. Kishimoto T. Furukawa A. Mori H. Tanaka Y. Dohmae N. et al.Crystal structure of Escherichia coli YidC, a membrane protein chaperone and insertase.Sci. Rep. 2014; 4: 7299Crossref PubMed Scopus (96) Google Scholar, 14Tanaka Y. Izumioka A. Abdul Hamid A. Fujii A. Haruyama T. Furukawa A. et al.2.8-Å crystal structure of Escherichia coli YidC revealing all core regions, including flexible C2 loop.Biochem. Biophys. Res. Commun. 2018; 505: 141-145Crossref PubMed Scopus (13) Google Scholar). It has been proposed that membrane protein TM segments are transferred from the Sec translocon into the lipid phase of the IM via the lateral gate formed between TM2 and TM7 of SecY (1Van den Berg B. Clemons W.M.J. Collinson I. Modis Y. Hartmann E. Harrison S.C. et al.X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Crossref PubMed Scopus (1004) Google Scholar, 15Kater L. Frieg B. Berninghausen O. Gohlke H. Beckmann R. Kedrov A. Partially inserted nascent chain unzips the lateral gate of the Sec translocon.EMBO Rep. 2019; 20e48191Crossref PubMed Scopus (27) Google Scholar). In contrast to a wealth of accumulated knowledge about structure and function of the Sec translocon and the motor proteins, only a limited information regarding functional relationships between these Sec components and other Sec-related factors that could contribute to maturation process of secretory proteins is available. During and/or after the translocation of proteins through the Sec translocon, the proper folding of periplasmic proteins and the efficient targeting of outer membrane proteins (OMPs) are regulated by many periplasmic chaperones, including SurA (a peptidyl–prolyl cis–trans isomerase, PPIase), Skp, DegP, and PpiD (16Stull F. Betton J.-M. Bardwell J.C.A. Periplasmic chaperones and prolyl isomerases.EcoSal Plus. 2018; https://doi.org/10.1128/ecosalplus.ESP-0005-2018Crossref PubMed Google Scholar, 17Sklar J.G. Wu T. Kahne D. Silhavy T.J. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli.Genes Dev. 2007; 21: 2473-2484Crossref PubMed Scopus (356) Google Scholar, 18Wang X. Peterson J.H. Bernstein H.D. Bacterial outer membrane proteins are targeted to the Bam complex by two parallel mechanisms.MBio. 2021; https://doi.org/10.1128/mBio.00597-21Crossref Scopus (13) Google Scholar). Among these chaperones, PpiD is unique in that it is a membrane protein that associates with the IM via a single N-terminal TM segment, whereas its large periplasmic C-terminal domain contains a parvulin-like PPIase domain, similar to SurA (19Dartigalongue C. Raina S. A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli.EMBO J. 1998; 17: 3968-3980Crossref PubMed Scopus (192) Google Scholar). The ppiD gene was first identified as a multicopy suppressor of a surA null mutation, and it was reported that ppiD deletion significantly decreases cellular OMP levels, whereas the ppiD null mutation causes synthetic lethality with the surA null mutation (19Dartigalongue C. Raina S. A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli.EMBO J. 1998; 17: 3968-3980Crossref PubMed Scopus (192) Google Scholar). It was therefore concluded that PpiD plays an important role in OMP biogenesis; however, several later studies were unable to reproduce published data supporting the functional importance of PpiD in OMP biogenesis (20Weininger U. Jakob R.P. Kovermann M. Balbach J. Schmid F.X. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity.Protein Sci. 2010; 19: 6-18Crossref PubMed Scopus (31) Google Scholar, 21Matern Y. Barion B. Behrens-Kneip S. PpiD is a player in the network of periplasmic chaperones in Escherichia coli.BMC Microbiol. 2010; 10: 251Crossref PubMed Scopus (47) Google Scholar, 22Justice S.S. Hunstad D.A. Harper J.R. Duguay A.R. Pinkner J.S. Bann J. et al.Periplasmic peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli.J. Bacteriol. 2005; 187: 7680-7686Crossref PubMed Scopus (120) Google Scholar). These studies instead reported genetic and physical interactions between PpiD and other periplasmic chaperones (SurA, Skp, and DegP), suggesting that PpiD participates in the periplasmic chaperone network and functions mainly to facilitate the early periplasmic folding of newly translocated proteins (21Matern Y. Barion B. Behrens-Kneip S. PpiD is a player in the network of periplasmic chaperones in Escherichia coli.BMC Microbiol. 2010; 10: 251Crossref PubMed Scopus (47) Google Scholar, 22Justice S.S. Hunstad D.A. Harper J.R. Duguay A.R. Pinkner J.S. Bann J. et al.Periplasmic peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for pilus biogenesis in Escherichia coli.J. Bacteriol. 2005; 187: 7680-7686Crossref PubMed Scopus (120) Google Scholar). In vitro studies have also suggested that PpiD plays a role in protein translocation mediated by the Sec translocon by interacting with SecY/E/G and nascent polypeptides emerging from the translocon (23Antonoaea R. Fürst M. Nishiyama K.-I. Müller M. The periplasmic chaperone PpiD interacts with secretory proteins exiting from the SecYEG translocon.Biochemistry. 2008; 47: 5649-5656Crossref PubMed Scopus (52) Google Scholar). Detailed photocrosslinking analyses have suggested that PpiD contacts the lateral gate region of SecY (24Sachelaru I. Petriman N.-A. Kudva R. Koch H.-G. Dynamic interaction of the Sec translocon with the chaperone PpiD.J. Biol. Chem. 2014; 289: 21706-21715Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and that the periplasmic domain of PpiD is located close to SecG (25Fürst M. Zhou Y. Merfort J. Müller M. Involvement of PpiD in Sec-dependent protein translocation.Biochim. Biophys. Acta. Mol. Cell Res. 2018; 1865: 273-280Crossref PubMed Scopus (15) Google Scholar). Recently, we used the pulse-chase and in vivo photocrosslinking experiment (PiXie) method (26Miyazaki R. Myougo N. Mori H. Akiyama Y. A photo-cross-linking approach to monitor folding and assembly of newly synthesized proteins in a living cell.J. Biol. Chem. 2018; 293: 677-686Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) to demonstrate that PpiD interacts directly with the nascent translocating Vibrio protein export monitoring polypeptide (VemP) (27Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (43) Google Scholar), which undergoes regulated translation elongation arrest in response to decreased cellular protein translocation activity (28Miyazaki R. Akiyama Y. Mori H. Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation.eLife. 2020; https://doi.org/10.7554/eLife.62623Crossref Google Scholar). Our in vivo studies have also indicated that physical interaction and cooperation between PpiD and SecD/F are required for the efficient translocation and translation arrest-cancellation of VemP. Based on these observations, we proposed that PpiD stimulates the forward movement of polypeptide substrates through the Sec translocon by capturing the substrate and transferring it to SecD/F and/or other periplasmic chaperones during the later stages of translocation (28Miyazaki R. Akiyama Y. Mori H. Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation.eLife. 2020; https://doi.org/10.7554/eLife.62623Crossref Google Scholar). YfgM and PpiD share the same type II (NIN–COUT) topology; they integrate into the IM via N-terminal TM segments, whereas the large C-terminal domain is exposed to the periplasm. The YfgM periplasmic domain is composed of four tetratricopeptide repeat (TPR) motifs, and TPR motif–containing domains (TPR domains) generally act as scaffolds to mediate protein–protein interactions (29Zeytuni N. Zarivach R. Structural and functional discussion of the tetra-trico-peptide repeat, a protein interaction module.Structure. 2012; 20: 397-405Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Several studies have suggested that PpiD forms a stable complex with YfgM (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 31Carlson M.L. Stacey R.G. Young J.W. Wason I.S. Zhao Z. Rattray D.G. et al.Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries.eLife. 2019; https://doi.org/10.7554/eLife.46615Crossref Scopus (42) Google Scholar, 32Maddalo G. Stenberg-Bruzell F. Götzke H. Toddo S. Björkholm P. Eriksson H. et al.Systematic analysis of native membrane protein complexes in Escherichia coli.J. Proteome Res. 2011; 10: 1848-1859Crossref PubMed Scopus (48) Google Scholar) that interacts with the Sec translocon (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), indicating that the function of YfgM is related to that of PpiD. Although yfgM gene deletion has been shown to induce cell envelope stress responses (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the physiological role of YfgM in protein translocation remains unclear, and the physiological significance and the molecular basis of the PpiD–YfgM interaction are unresolved. In this study, we conducted genetic and biochemical studies using yfgM and ppiD mutant strains and found that YfgM stabilizes PpiD and is involved in VemP translocation. In addition, we investigated the modes of interaction between YfgM and PpiD and the Sec translocon through p-benzoyl-l-phenylalanine (pBPA)-mediated in vivo photocrosslinking analyses (33Miyazaki R. Akiyama Y. Mori H. A photo-cross-linking approach to monitor protein dynamics in living cells.Biochim. Biophys. Acta Gen. Subj. 2020; 1864129317Crossref PubMed Scopus (15) Google Scholar, 34Chin J.W. Schultz P.G. In vivo photocrosslinking with unnatural amino acid mutagenesis.ChemBioChem. 2002; 3: 1135-1137Crossref PubMed Scopus (127) Google Scholar) targeted to YfgM, PpiD, and SecG. Based on these results, we discuss the possible physiological significance of YfgM–PpiD complex formation. Previous studies have shown that YfgM forms a stable complex with PpiD (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 31Carlson M.L. Stacey R.G. Young J.W. Wason I.S. Zhao Z. Rattray D.G. et al.Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries.eLife. 2019; https://doi.org/10.7554/eLife.46615Crossref Scopus (42) Google Scholar, 32Maddalo G. Stenberg-Bruzell F. Götzke H. Toddo S. Björkholm P. Eriksson H. et al.Systematic analysis of native membrane protein complexes in Escherichia coli.J. Proteome Res. 2011; 10: 1848-1859Crossref PubMed Scopus (48) Google Scholar). Therefore, we first examined whether YfgM affects the stability of PpiD in Escherichia coli using a strain lacking the yfgM gene. Immunoblot analyses of total cellular proteins using anti-PpiD antibodies showed that the absence of YfgM exerted no detectable effect on the cellular levels of the full-length (FL) PpiD protein, as reported previously (Fig. 1A, top panel; (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar)). However, we noticed that a faint band (hereafter denoted as PpiD′) migrated slightly faster than FL PpiD in the ΔyfgM strain (Fig. 1A, top and upper-middle panels). To examine whether PpiD′ was derived from FL PpiD, we monitored the stability of fully synthesized PpiD after blocking cellular de novo protein synthesis using spectinomycin (Spc; Spc-chase experiment). The amount of PpiD′ gradually increased with a concomitant decrease in the levels of FL PpiD during the Spc-chase period (Fig. S1), strongly suggesting that PpiD′ is a degradation product of FL PpiD. In cells chromosomally expressing a PpiD derivative with a C-terminal His10-tag (PpiD-His10), the degradation product was detected with anti-PpiD but not with anti-His antibodies, even after prolonged exposure during immunoblot visualization (Fig. 1B, bottom panel). In addition, ectopic YfgM-His10 expression in the ΔyfgM strain suppressed the generation of PpiD' (Fig. 1, B and C). Taken together, these results suggest that a C-terminal portion of PpiD becomes susceptible to degradation by one or more unidentified proteases in the absence of YfgM (Fig. 1B, schematic picture). It has been reported that the yfgM gene contains a σE-dependent promotor for the downstream bamB gene, which encodes a component of the β-barrel assembly machinery (BAM) complex that is crucial for OMP biogenesis (Fig. 1D) (35Rhodius V.A. Suh W.C. Nonaka G. West J. Gross C.A. Conserved and variable functions of the sigmaE stress response in related genomes.PLoS Biol. 2006; 4: e2Crossref PubMed Scopus (427) Google Scholar). Consistently, we found that BamB accumulation levels were appreciably lower in our yfgM deletion strain than in the wildtype strain (Fig. 1A, bottom panel), indicating that the observed decrease in BamB levels is somehow involved in PpiD′ generation through the perturbation of OMP biogenesis. Therefore, we constructed a yfgM mutant strain carrying a mutant gene (yfgM(TM f.s.)) that expresses a nonfunctional YfgM variant (YfgM(TM F.S.)) but retains the intact σE-dependent promotor (Fig. 1D). Within the YfgM(TM F.S.) protein, the hydrophobic amino acid sequence for the TM segment of YfgM (Ala-24 to Asn-43) was replaced by a completely different hydrophilic sequence because of frameshift mutations (Fig. S2). Consequently, YfgM(TM F.S.) did not accumulate in cells, likely as it was not targeted to the IM (Fig. 1A, lower-middle panel). Although BamB accumulation was comparable in the yfgM(TM f.s.) and wildtype strains, as expected (Fig. 1A, bottom panel), PpiD′ was still detected as in the ΔyfgM strain (Fig. 1A) and YfgM-His10 overproduction from a plasmid suppressed PpiD′ generation (Fig. 1C). These results indicate that the absence of YfgM is the main reason for the generation of PpiD′ and strongly suggest that YfgM stabilizes PpiD, presumably by forming a YfgM–PpiD complex. Since PpiD, the partner protein of YfgM, plays a crucial role in the translocation-coupled translation arrest-cancellation (TTAC) of VemP (28Miyazaki R. Akiyama Y. Mori H. Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation.eLife. 2020; https://doi.org/10.7554/eLife.62623Crossref Google Scholar) and the PpiD–YfgM complex is known to interact with the Sec translocon (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), we next investigated the possible role of YfgM in the maturation (translocation) of VemP. To this end, we examined the effect of yfgM deletion on the TTAC kinetics of VemP using a model substrate, VemP-3xFLAG-Myc (VemP-F3M; a VemP derivative containing a 3xFLAG tag and a Myc tag at its C terminus). The addition of these tags allowed the three VemP-derived species to be discriminated using SDS-PAGE: the arrested product with an unprocessed signal sequence (AP-unpro), the arrested product lacking the signal sequence (AP-pro), and the FL mature product (FL-mature; Fig. 2A) (36Mori H. Sakashita S. Ito J. Ishii E. Akiyama Y. Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.J. Biol. Chem. 2018; 293: 2915-2926Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We expressed VemP-F3M from a plasmid in E. coli and examined its behavior using pulse-chase experiments. Consistent with previous results (36Mori H. Sakashita S. Ito J. Ishii E. Akiyama Y. Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.J. Biol. Chem. 2018; 293: 2915-2926Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), the arrested VemP products (AP-unpro and AP-pro) were gradually converted into FL-mature in the wildtype strain, whereas this process was obviously retarded in the ppiD and yfgM deletion strains, indicating that the TTAC of VemP was slowed (Figs. 2A and S3A). The translation-arrested VemP state was also stabilized in the yfgM(TM f.s.) mutant strain (Figs. 2B and S3B), and YfgM expression from a plasmid canceled the stabilization of the arrested VemP state in the yfgM deletion strain (Fig. S4). Importantly, the ΔyfgM mutation had no additive or synergistic effect on the stabilization of the arrested VemP state (Figs. 2A and S3A) when introduced into the ΔppiD mutant strain, suggesting that YfgM and PpiD act at the same step. By contrast, all these mutations examined only slightly retarded signal sequence cleavage of maltose-binding protein (MBP), a Sec-dependent secretory protein (Figs. 2, A, B, S3, A, and B), showing that both PpiD and YfgM do not play crucial roles in the initial step of the MBP export before its signal peptide processing. The effects of ppiD and yfgM deletion on the TTAC of VemP were confirmed using a VemP–PhoA reporter assay (Fig. S5A). The wildtype strain with a plasmid carrying the vemP–phoA fusion gene displayed significant PhoA activity as the FL VemP–PhoA fusion protein was translated and exported into the periplasmic space because of effective VemP arrest-cancellation in this strain. Conversely, the relative PhoA activity in the ΔyfgM and the ΔppiD mutant strains expressing VemP–PhoA was about half of that in the wildtype strain expressing VemP–PhoA, suggesting that the translation-arrested VemP state was more stabilized in these mutant strains than in the wildtype strain. Again, no additive or synergistic effects were observed in the double mutant strain. Together, these results indicate that YfgM and PpiD likely affect the TTAC of VemP by forming a functional complex. Since VemP monitors cellular protein export activity and regulates V.secD2/F2 gene expression via translation arrest in Vibrio alginolyticus (27Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (43) Google Scholar), we examined the effect of yfgM deletion on VemP translation-arrest–mediated V.SecD2 expression in V. alginolyticus. In Na+-rich medium, V.SecD2 expression was strongly repressed because V.SecD1/F1 was fully functional, leading to efficient VemP arrest-cancellation (27Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (43) Google Scholar). In contrast, V.SecD2 accumulation increased in the yfgM deletion V. alginolyticus strain as well as in the ΔV.secD1/F1 and ΔV.ppiD strains (Fig. 2C, middle panel) (27Ishii E. Chiba S. Hashimoto N. Kojima S. Homma M. Ito K. et al.Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E5513-E5522Crossref PubMed Scopus (43) Google Scholar). In addition, a faster-migrating band similar to E. coli PpiD′, presumably representing a V.PpiD degradation product, was detected in the ΔV.yfgM strain (Fig. 2C, bottom panel). These results suggest that YfgM plays a role alongside PpiD in VemP translation arrest-cancellation and V.SecD2 repression under Na+-rich conditions in Vibrio cells. Although YfgM is known to interact with PpiD to form a stable complex that interacts with the Sec translocon (30Götzke H. Palombo I. Muheim C. Perrody E. Genevaux P. Kudva R. et al.YfgM is an ancillary subunit of the SecYEG translocon in Escherichia coli.J. Biol. Chem. 2014; 289: 19089-19097Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), the molecular details of these interactions are currently unknown; therefore, we conducted a systematic photocrosslinking analysis to elucidate how YfgM interacts with PpiD and SecY/E/G. We constructed 40 different YfgM(pBPA)-His10 derivatives by systematically introducing pBPA into every fifth residue in YfgM. Total cellular proteins in UV-irradiated cells expressing YfgM(pBPA)-His10 were analyzed using immunoblotting with anti-His, anti-PpiD, and anti-SecG antibodies (Fig. S6). We observed six possible crosslinked products that crossreacted with anti-PpiD antibodies (YfgM derivatives with pBPA at Tyr-86, Val-126, Val-136, Trp-156, Trp-181, or Met-196) and three possible crosslinked products that crossreacted with anti-SecG antibodies (YfgM derivatives with pBPA at Asn-6, Val-11, or Glu-66). Although some of these products were not clearly detected with anti-His antibodies, all were successfully purified using nickel–nitrilotriacetic acid agarose (Fig. 3, A and B), confirming that they were indeed YfgMxPpiD or YfgMxSecG crosslinked products. The YfgM derivatives with pBPA at Trp-181 or Met-196 generated two cros

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