TABLE 5 - uploaded by Kit Pogliano
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The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromoso...
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... which is less hydrophobic than other regions containing membrane-spanning segments (Fig. 6). The first region was shown not to be a stable membrane-spanning segment by the high specific activities, and therefore external locations, of fusions both before (D419) and after (D445) it (Table 5). The second region was shown to contain at least one membrane-spanning segment by the high specific activity of fusion D588, demonstrating that it was exported and thus preceded by a membrane-spanning segment. ...
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... other observations support this conclusion. First, TnphoA insertions in secD and secF produce similar units of alkaline phosphatase activity in both high-and low-copy-number plasmids (Tables 5 and 6). The changes in activity observed upon reducing the copy number of the fusions are consistent with the effect of the pcnB mutation (26) and with estimates of the copy numbers of pBR322-based plasmids (25). ...
Citations
... In E. coli formt SecDF einen stabilen Komplex mit dem nicht essentiellen YajC (Pogliano & Beckwith, 1994b;Tsukazaki et al., 2011). YajC ist nur 11,9 kDa (110 Aminosäuren) groß und besitzt nur eine einzige Transmembrandomäne (UniProt P0ADZ7). ...
... Die Funktion von YajC ist bisher ungeklärt. Es ist in E. coli nicht essentiell (Pogliano & Beckwith, 1994b) und ist nicht in allen Prokaryonten vorhanden (Crane & Randall, 2017). Da YajC in einem 5-10-fachen Überschuss zu SecDF vorkommt (Pogliano & Beckwith, 1994b), erfüllt es möglicherweise Aufgaben unabhängig vom Proteinexport (Crane & Randall, 2017). ...
... Es ist in E. coli nicht essentiell (Pogliano & Beckwith, 1994b) und ist nicht in allen Prokaryonten vorhanden (Crane & Randall, 2017). Da YajC in einem 5-10-fachen Überschuss zu SecDF vorkommt (Pogliano & Beckwith, 1994b), erfüllt es möglicherweise Aufgaben unabhängig vom Proteinexport (Crane & Randall, 2017). ...
YidC/Oxa1/Alb3-Insertasen und das Sec-Translokon sind über alle drei Reiche des Lebens konserviert und stellen den wichtigsten Weg für integrale Proteine in Zellmembranen und Membranen von eukaryontischen Organellen dar. Die Insertion von Membranproteinen in die innere Membran von Gram-negativen Bakterien erfolgt hauptsächlich über die SecYEG-Translokase und die YidC-Insertase, die unabhängig voneinander oder in Kooperation miteinander agieren können. Für die kooperative Insertion wird ein enger Kontakt zwischen SecY und YidC vorausgesetzt. Aufgrund von früheren Interaktions-Studien und einer kürzlich gelösten Struktur des sogenannten Holotranslokons mit geringer Auflösung (14 Å) wird von einem Kontakt zwischen dem lateral gate von SecY und der hydrophoben Substratrutsche von YidC ausgegangen. Welche konkreten Domänen von YidC und SecY dabei direkt miteinander interagieren, war bisher nicht bekannt. Ziel dieser Arbeit war es, den Kontakt zwischen SecY und YidC detaillierter zu beschreiben. In vitro wurde über FRET-Messungen mit Fluoreszenzfarbstoff-markiertem SecY und YidC eine hohe Affinität für die Interaktion der beiden Proteine sowohl in Detergens als auch in DOPC-Proteoliposomen ermittelt. Für die Stöchiometrie der SecY/YidC-Interaktion wurde der Faktor eins bestimmt. Um die genauen Kontakte zwischen SecY und YidC zu identifizieren, wurden in vivo Disulfid-Cross-Linking-Experimente durchgeführt. Es konnten direkte Kontakte zwischen der TM3 und TM8 des SecY lateral gates und der TM3 beziehungsweise TM5 der hydrophoben Substratrutsche von YidC gezeigt werden. Zudem wurde eine YidC-Mutante mit fünf Serin-Substitutionen, die den Wachstumsdefekt eines YidC-Depletionsstamms nicht wiederherstellen konnte, charakterisiert. Obwohl die Serin-Positionen alle in der Mitte und der periplasmatischen Hälfte der hydrophoben Rutsche von YidC liegen und vier der Positionen mit Substrat-Kontaktstellen übereinstimmen, konnte keine Inhibierung der Insertion der YidC-abhängigen Substrate M13 procoat und Pf3 coat durch die 5S Mutante im Vergleich zum Wildtyp beobachtet werden. Für die YidC-only Insertion scheint ein Minimum an Hydrophobizität nötig zu sein, das bei dieser Mutante nicht unterschritten wird. In vitro FRET-Experimente zeigten eine gestörte Interaktion zwischen SecY und dieser YidC 5S Mutante und bestätigten damit erneut eine Beteiligung der hydrophoben Rutsche am SecY/YidC-Kontakt. Anhand der ermittelten Cross-Link-Kontakte und der Ergebnisse der FRET-Messungen konnte ein mögliches Kontaktmodell erstellt werden, in dem das SecY lateral gate der hydrophoben Substrat-Rutsche und der sich zwischen TM3 und TM5 öffnenden hydrophilen Tasche von YidC gegenüber steht, wodurch sich eine gemeinsame SecY/YidC-Pore bildet. Die vorliegende Arbeit liefert damit weitere Beweise, dass das lateral gate der Sec-Translokase direkt mit der hydrophoben Substratrutsche von YidC interagiert. In einem weiteren Projekt wurde ein SecY-YidC-Fusionsprotein zur Sicherstellung eines engen Kontakts, der korrekten Orientierung der Proteine zueinander und der richtigen Stöchiometrie in rekonstituierten Proteoliposomen kloniert. Für eine gemeinsame Studie mit der ETH Zürich wurden Proteoliposomen, die das Fusionsprotein, SecYEG, YidC oder SecYEG zusammen mit YidC enthielten, von mir in Hohenheim hergestellt. Die schrittweise Insertion des Sec/YidC-abhängigen Substrats LacY in diese Proteoliposomen wurde von einer Arbeitsgruppe der ETH Zürich mithilfe der AFM-basierten Einzelmolekül-Kraftspektroskopie beobachtet. Die LacY-Insertion wird im Falle des Fusionsproteins und SecYEG in Kombination mit YidC von der Sec-Translokase dominiert, während YidC vermutlich nur eine unterstützende Rolle bei der Proteinfaltung einnimmt.
... While biochemical characterizations indicate a very low copy number (approx. 50 copies per E. coli cell (88,89), ribosome profiling data suggest more than 2500 copies (54). If SecDF is indeed required for a stable YidC-SecYEG complex, SecDF would limit the amount of detectable SecYEG-YidC complexes, if substoichiometric to SecYEG. ...
... ratio of SecYHisEG/SecYEG (His/Ctl), the known or predicted function and the cellular copy number of the protein in E. coli, based on ribosome profiling (54). ↑ indicates that the copy number based on ribosome profiling is significantly higher than the copy number based on other methods (89). ...
The SecYEG translocon constitutes the major protein transport channel in bacteria and transfers an enormous variety of different secretory and inner-membrane proteins. The minimal core of the SecYEG translocon consists of three inner-membrane proteins, SecY, SecE, and SecG, which, together with appropriate targeting factors, are sufficient for protein transport in vitro. However, in vivo the SecYEG translocon has been shown to associate with multiple partner proteins, likely allowing the SecYEG translocon to process its diverse substrates. To obtain a global view on SecYEG plasticity in Escherichia coli, here we performed a quantitative interaction proteomic analysis, which identified several known SecYEG-interacting proteins, verified the interaction of SecYEG with quality-control proteins, and revealed several previously unknown putative SecYEG interacting proteins. Surprisingly, we found that the chaperone complex PpiD/YfgM is the most prominent interaction partner of SecYEG. Detailed analyses of the PpiD–SecY interaction by site-directed cross-linking revealed that PpiD and the established SecY partner protein YidC use almost completely overlapping binding sites on SecY. Both PpiD and YidC contacted the lateral gate, the plug domain, and the periplasmic cavity of SecY. However, quantitative MS and cross-linking analyses revealed that despite having almost identical binding sites, their binding to SecY is non-competitive. This observation suggests that the SecYEG translocon forms different substrate-independent subassemblies in which SecYEG either associates with YidC or with the PpiD/YfgM complex. In summary, the results of this study indicate that the PpiD/YfgM chaperone complex is a primary interaction partner of the SecYEG translocon.
... SecDF plays a role in the downstream stages of translocation of secretory proteins as well as in membrane protein biogenesis and stabilization of the SecY protein forming the pore (4,26,27,66). Structure elucidation showed that the SecDF complex consists of 12 TMDs, 6 TMDs per protein, and 6 periplasmic domains (P1-P6) (Figure 5a) (79,86). The transmembrane domains of SecD and SecF form a base substructure that is structurally homologous (102). ...
... This change possibly indicates a disassembly of the holotranslocon, as the function of SecDF depends on the PMF. Furthermore, biochemical, genetic, and single-molecule observations indicate a low abundance of the SecDF complex (67,79). Studies on the stoichiometry of the holotranslocon suggest that a single SecDF complex associates with a monomeric SecYEG translocon (92). ...
Single-molecule studies provide unprecedented details about processes that are difficult to grasp by bulk biochemical assays that yield ensemble-averaged results. One of these processes is the translocation and insertion of proteins across and into the bacterial cytoplasmic membrane. This process is facilitated by the universally conserved secretion (Sec) system, a multi-subunit membrane protein complex that consists of dissociable cytoplasmic targeting components, a molecular motor, a protein-conducting membrane pore, and accessory membrane proteins. Here, we review recent insights into the mechanisms of protein translocation and membrane protein insertion from single-molecule studies.
... ATP hydrolysis resets the 2HF without pulling on the polypeptide chain. affecting each of these components can cause defects in SecAmediated protein translocation in vivo (Gardel et al. 1987(Gardel et al. , 1990Pogliano and Beckwith 1994;Samuelson et al. 2000), suggesting that the holotranslocon or its individual constituents assist in SecA-mediated translocation. ...
... In E. coli, YajC is encoded in the same polycistronic message as SecD and -F and appears to interact with the SecDF complex (Pogliano and Beckwith 1994;Duong and Wickner 1997). However, the role of YajC in SecA-mediated protein translocation is unknown. ...
... However, the role of YajC in SecA-mediated protein translocation is unknown. However, mutations in the yajC gene do not cause a detectable translocation defect on their own (Pogliano and Beckwith 1994). ...
In bacteria, translocation of most soluble secreted proteins (and outer membrane proteins in Gram-negative bacteria) across the cytoplasmic membrane by the Sec machinery is mediated by the essential ATPase SecA. At its core, this machinery consists of SecA and the integral membrane proteins SecYEG, which form a protein conducting channel in the membrane. Proteins are recognised by the Sec machinery by virtue of an internally encoded targeting signal, which usually takes the form of an N-terminal signal sequence. In addition, substrate proteins must be maintained in an unfolded conformation in the cytoplasm, prior to translocation, in order to be competent for translocation through SecYEG. Recognition of substrate proteins occurs via SecA-either through direct recognition by SecA or through secondary recognition by a molecular chaperone that delivers proteins to SecA. Substrate proteins are then screened for the presence of a functional signal sequence by SecYEG. Proteins with functional signal sequences are translocated across the membrane in an ATP-dependent fashion. The current research investigating each of these steps is reviewed here.
... The yajC gene, which encodes an inner membrane protein that is part of the Sec protein translocase complex (Pogliano & Beckwith, 1994b), was found here to be essential for P22 proliferation in several experiments (Table 1). The fraction of bacteria with Tn5 integrations in yajC increased upon challenge with P22 (Table 1), and no plaques were observed in spot tests ( Figure 5) when P22 was plated on the yajC deleted strain STM14_0481 from the SGD panel (Porwollik et al., 2014). ...
Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of <0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2,000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane. This article is protected by copyright. All rights reserved.
... VemP translation undergoes elongation arrest near its C-terminus (6). To distinguish between the fully synthesized VemP polypeptide and the arrested polypeptide by SDS-PAGE after removal of the tRNA moiety, we constructed a plasmid, pTS47, carrying vemP-(flag) 3 Bands corresponding to the arrested polypeptide with the unprocessed signal sequence (156 residues), the arrested and signal sequence-processed polypeptide (130 residues), and the mature (signal sequenceprocessed) full-length product (174 residues) were clearly separated by large differences in mobility on SDS-PAGE (Fig. 1B, compare lanes 1-3 and 10-12). ...
... stability of the elongation-arrested form of VemP by pulse-chase experiments using E. coli expressing the VemP-(FLAG)3 -Myc fusion protein. We observed that the arrested/unprocessed form of VemP remained stable when the translocation machinery was inactivated (Figs. ...
VemP (
V
ibrioproteinexportmonitoringpolypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor inVibrioand regulates expression of the downstreamV.secDF2genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156codon ofvemPencounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor thein vivoarrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met120-Phe137) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.
... YajC is a small, single-spanning membrane protein encoded upstream of SecD in the yajC-secDF operon. Its expression level is tenfold higher than that of SecDF 23 , which might explain the presence of YajC complexes that do not contain SecDF 9, 24 . SecDFYajC was cross-linked to SecG 63 ) and the SecY channel interior and pore ring (right panel). ...
... This discrepancy is probably explained by the fact that we used a conditional SecDF depletion strain, while SecDF-overexpressing strains were often analyzed for a possible role of SecDF on SecY-YidC interaction 10,25 . Intriguingly, the overexpression of SecDF induces a prlA4 phenotype 11,23 , which is connected with a weakened association between the subunits of the SecYEG translocon 42,43 and a SecYEG mediated ion leak 44 . We therefore analyzed whether the SecY-YidC interaction was influenced by the prlA4 allele of SecY. ...
The heterotrimeric SecYEG complex cooperates with YidC to facilitate membrane protein insertion by an unknown mechanism. Here we show that YidC contacts the interior of the SecY channel resulting in a ligand-activated and voltage-dependent complex with distinct ion channel characteristics. The SecYEG pore diameter decreases from 8 Å to only 5 Å for the YidC-SecYEG pore, indicating a reduction in channel cross-section by YidC intercalation. In the presence of a substrate, YidC relocates to the rim of the pore as indicated by increased pore diameter and loss of YidC crosslinks to the channel interior. Changing the surface charge of the pore by incorporating YidC into the channel wall increases the anion selectivity, and the accompanying change in wall hydrophobicity is liable to alter the partition of helices from the pore into the membrane. This could explain how the exit of transmembrane domains from the SecY channel is facilitated by YidC.
... SecD and SecF possess a very large first periplasmic domain that is important for catalyzing protein translocation ( Nouwen, Piwowarek, Berrelkamp, & Driessen, 2005). Cells lacking SecD and/or SecF have a severe export defect and are barely viable ( Pogliano & Beckwith, 1994a, 1994b). YidC provides new mechanistic insights of how transmembrane proteins achieve the transition from an aqueous environment in the cytoplasm to the hydrophobic lipid bilayer environment of the membrane ( Kuhn & Kiefer, 2016). ...
Vibrio alginolyticus caused great losses to aquaculture. Adhesion is an important virulence factor of V. alginolyticus. In this study, the relationship between V. alginolyticus adhesion and type II secretion system genes (secA, secD, secF, yajC, and yidC) was determined using gene silencing, qRT-PCR and in vitro adhesion assay. The results showed that the expression of target genes and the bacterial adhesion exhibited significant decreases after transient gene silencing and stable gene silencing, which indicated that secA, secD, secF, yajC, and yidC played roles in the bacterial adhesion of V. alginolyticus. The expression of secA, secD, secF, yajC, and yidC were significantly influenced by temperature, salinity, pH and starvation. The results indicated that the expression of secA, secD, secF, yajC, and yidC were sensitive to different environmental factors, whereas environmental factors can affect V. alginolyticus adhesion via the expression of secA, secD, secF, yajC, and yidC.
... b [33] Determined from measurement of alkaline phosphatase activity of a secE-phoA fusion. d [34] Determined from measurement of alkaline phosphatase activity of a secD-phoA fusion. e [30] Determined by mass spectrometry. ...
SecB, a small tetrameric chaperone in Escherichia coli, plays a crucial role during protein export via the general secretory pathway by binding precursor polypeptides in a nonnative conformation and passing them to SecA, the ATPase of the translocon. The dissociation constants for the interactions are known; however to relate studies in vitro to export in a living cell requires knowledge of the concentrations of the proteins in the cell. Presently in the literature there is no report of a rigorous determination of the intracellular concentration of SecB. The values available vary over 60 fold and the details of the techniques used are not given. Here we use quantitative immunoblotting to determine the level of SecB expressed from the chromosome in E.coli grown in two commonly used media. In rich medium SecB was present at 1.6 ± 0.2 μM and in minimal medium at 2.5 ± 0.6 μM. These values allow studies of SecB carried out in vitro to be applied to the situation in the cell as SecB interacts with its binding partners to move precursor polypeptides through the export pathway.
... In particular, SecDF slows down the back and forward movement of secreted proteins inside SecYEG channel (Nouwen et al. 2005). Consequently, the deletion of either SecD or SecF resulted in a severe defect in secretion in vivo (Pogliano and Beckwith 1994b), and a decrease of membrane-inserted SecA (Eichler and Wickner 1997). By contrast, overexpression of SecDF led an opposite effect (Kim et al. 1994;Pogliano and Beckwith 1994a). ...
... Indeed, not only SecDF acts together and has 12 transmembrane segments and two large periplasmic domains (Tsukazaki et al. 2011), but also their codes are located in a single open reading frame in many species (Bolhuis et al. 1998). In fact, there are only 10-30 copies of SecDF per cell, which are 10 times less abundant than SecYEG (Pogliano and Beckwith 1994b), so targeting of SecDF would not require a huge amount of antibacterial agents. Take a step further, it is necessary to decide which is the best candidate among SecDF, SecD, and SecF, for the reason that SecDF exists in separate forms (Zhou et al. 2014). ...
Staphylococcus aureus causes many infections and its drug resistance is a worrying challenge for medical care. The SecD subunit of Sec secretion system in methicillin-resistant S. aureus is an attractive target because SecD dysfunction leads to the death of bacteria and SecD as a target is more efficient than SecA and SecF. Evolution could have made SecD to become insensitive to antibacterial agents although the drugs directly against SecD have yet to develop. So far, no detailed information on SecD evolution has been available, thus 2686 SecD sequences with full taxonomic information from kingdom to species were analyzed. First, the variance of pairwise p-distance was evaluated for each taxonomic group. Second, the variance was further partitioned into intergroup and intragroup variances for quantification of horizontal and vertical gene transfer. Third, phylogenetic tree was built to trace the evolutionary pathway. The results showed that overall evolution of SecDs appears to have undergone horizontal and vertical gene transfer. Only 0.5% horizontal transfers were found between any two SecDs in S. aureus, 6.8% and 8.8% horizontal transfers were found between any two Staphylococcus SecDs from different and the same species, and only one SecD from S. aureus was located far away from its sister cluster. Thus, statistic and evolutionary analyses demonstrate that the SecDs from staphylococcus species have a small chance of mutating, and provide taxonomic evidence to use the SecD as a potential target for new generation of antibacterial agents against S. aureus.
