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Bacterial cells utilize monitoring substrates, which undergo force-sensitive translation elongation arrest, to feedback-regulate a Sec-related gene. Vibrio alginolyticus VemP controls the expression of SecD/F that stimulates a late step of translocation by undergoing export-regulated elongation arrest. Here, we attempted at delineating the pathway of the VemP nascent-chain interaction with Sec-related factors, and identified the signal recognition particle (SRP) and PpiD (a membrane-anchored periplasmic chaperone) in addition to other translocon components and a ribosomal protein as interacting partners. Our results showed that SRP is required for the membrane-targeting of VemP, whereas PpiD acts cooperatively with SecD/F in the translocation and arrest-cancelation of VemP. We also identified the conserved Arg-85 residue of VemP as a crucial element that confers PpiD-dependence to VemP and plays an essential role in the regulated arrest-cancelation. We propose a scheme of the arrest-cancelation processes of VemP, which likely monitors late steps in the protein translocation pathway., Competing Interests: RM, YA, HM No competing interests declared, (© 2020, Miyazaki et al.)

VemP ( V ibrio protein e xport m onitoring p olypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln 156 codon of vemP encounters 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 the in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His 138 -Phe 157 ) 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 (Met 120 -Phe 137 ) 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., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine-Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles.

Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-β-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases., (Copyright © 2013 Elsevier B.V. All rights reserved.)

When bound to ADP, ATP-dependent protease FtsH subunits adopt either an "open" or "closed" conformation. In the open state, the protease catalytic site is located in a narrow space covered by a lidlike helix. This space disappears in the closed form because the lid helix bends at Gly448. Here, we replaced Gly448 with various residues that stabilize helices. Most mutants retained low ATPase activity and bound to the substrate protein, but lost protease activity. However, a mutant proline substitution lost both activities. Our study shows that the conformational transition of the lid helix is essential for the function of FtsH.

Two membrane proteases, FtsH and HtpX, are jointly essential for Escherichia coli cell viability, presumably through their abilities to degrade abnormal membrane proteins. To search for additional cellular factors involved in membrane protein quality control, we isolated multicopy suppressors that alleviated the growth defect of the ftsH/htpX dual disruption mutant. One of them was ybbK, which is renamed qmcA, encoding a membrane-bound prohibitin homology (PHB) domain family protein. Multicopy suppression was also observed with hflK-hflC, encoding another set of PHB domain membrane proteins, which had been known to form a complex (HflKC) and to interact with FtsH. Whereas the DeltaftsH sfhC21 (a viability defect suppressor for DeltaftsH) strain exhibited temperature sensitivity in the presence of cAMP, additional disruption of both qmcA and hflK-hflC exaggerated the growth defect. Pull-down and sedimentation experiments showed that QmcA, like HflKC, forms an oligomer and interacts with FtsH. Protease accessibility assays revealed that QmcA, unlike periplasmically exposed HflKC, possesses a cytoplasmically disposed large C-terminal domain, thus assuming the type I (NOUT-CIN) orientation. We discuss possible significance of having PHB domains on both sides of the membrane.

Escherichia coli HtpX is a putative membrane-bound zinc metalloprotease that has been suggested to participate in the proteolytic quality control of membrane proteins in conjunction with FtsH, a membrane-bound and ATP-dependent protease. Here, we biochemically characterized HtpX and confirmed its proteolytic activities against membrane and soluble proteins. HtpX underwent self-degradation upon cell disruption or membrane solubilization. Consequently, we purified HtpX under denaturing conditions and then refolded it in the presence of a zinc chelator. When supplemented with Zn2+, the purified enzyme exhibited self-cleavage activity. In the presence of zinc, it also degraded casein and cleaved a solubilized membrane protein, SecY. We verified its ability to cleave SecY in vivo by overproducing both HtpX and SecY. These results showed that HtpX is a zinc-dependent endoprotease member of the membrane-localized proteolytic system in E. coli.

FtsH is a cytoplasmic membrane protein that has N-terminally located transmembrane segments and a main cytosolic region consisting of AAA-ATPase and Zn2+-metalloprotease domains. It forms a homo-hexamer, which is further complexed with an oligomer of the membrane-bound modulating factor HflKC. FtsH degrades a set of short-lived proteins, enabling cellular regulation at the level of protein stability. FtsH also degrades some misassembled membrane proteins, contributing to their quality maintenance. It is an energy-utilizing and processive endopeptidase with a special ability to dislocate membrane protein substrates out of the membrane, for which its own membrane-embedded nature is essential. We discuss structure-function relationships of this intriguing enzyme, including the way it recognizes the soluble and membrane-integrated substrates differentially, on the basis of the solved structure of the ATPase domain as well as extensive biochemical and genetic information accumulated in the past decade on this enzyme.

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.

FtsH is an ATP-dependent and membrane-associated protease, which exerts processive proteolysis against membrane-embedded and soluble substrate proteins. Although previous studies suggested that it functions as a homo-oligomer and it also interacts with HflK-HflC membrane protein complex (HflKC), it is still important to address the question of what kind of supramolecular assembly FtsH forms in wild-type cells. Now we show that FtsH in wild-type Escherichia coli cells exists exclusively as a large complex, termed FtsH holo-enzyme, which can be separated from bulk of membrane proteins after detergent solubilization and velocity sedimentation. This complex appears to have molecular mass of around 1000 kDa. A tentative model is presented that it is composed of hexamers of FtsH and of HflKC, with an ability to bind one or a few substrate molecules.

Escherichia coli FtsH is a membrane-bound and ATP-dependent protease responsible for degradation of several membrane proteins. The FtsH action is processive and presumably involves dislocation of the substrate from the membrane to the cytosol. Although elucidation of its molecular mechanism requires an in vitro reaction system, in vitro activities of this enzyme against membrane protein substrates have only been assayed using detergent-solubilized components. Here we report on the construction of in vitro reaction systems for FtsH-catalyzed membrane protein degradation. A combination of two inverted membrane vesicles or of two proteoliposomes, one bearing the enzyme and the other bearing a substrate, was fused by polyethylene glycol 3350 treatment. Addition of ATP then resulted in degradation of the substrate. It was shown that FtsH can function in the process of membrane proteins degradation without aid from any other cellular factors.

The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation. As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the secY39 defect. A specific secE mutation, designated secE105, was thus isolated. This mutation proved to be identical with the prlG2 mutation and to suppress a number of cold-sensitive secY mutations. However, other prlG mutations did not effectively suppress the secY defects. Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association. In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation. It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.

FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.

Background: The abnormal accumulation of misfolded proteins outside the plasma (cytoplasmic or inner) membrane up-regulates the synthesis of a class of envelope-localized catalysts of protein folding and degradation. The pathway for this transmembrane signalling is mediated by the CpxR-CpxA two-component phospho-relay mechanism., Results: We now show that an abnormality in the plasma membrane proteins, due either to the impairment of FtsH, a protease acting against integral membrane proteins, or to the overproduction of a substrate membrane protein of FtsH, activates this stress response pathway. Under such conditions, the cpxR gene function becomes essential for cell growth. We further show that the expression of a putative protease, HtpX, in the plasma membrane, is under the control of CpxR. Synthetic growth inhibition was observed when the ftsH and htpX disruption mutations had been combined, suggesting that these gene products have some complementary or overlapping proteolytic functions. Topology analyses indicated that the metalloproteinase active site of HtpX is located on the cytosolic side of the membrane., Conclusions: Taken together, these results suggest that the Cpx "extracytoplasmic" stress response system controls the quality of the plasma membrane, even on its cytoplasmic side.

FtsH is a membrane-bound, ATP-dependent metalloprotease in Escherichia coli that degrades some integral membrane proteins and cytoplasmic proteins. In this study, we show that FtsH-dependent degradation of both membrane-bound and soluble proteins is retarded when cells are treated with carbonyl cyanide-3-chlorophenylhydrazone or 2,4-dinitrophenol uncouplers, which dissipate the proton-motive force. In vitro casein degradation by membrane-integrated FtsH was stimulated by succinate, a respiratory substrate; this stimulation was counteracted by cyanide-3-chlorophenylhydrazone. Potassium thiocyanate, which specifically collapses Deltapsi, partially canceled the effect of succinate, but ammonium sulfate, which collapses DeltapH, showed little effect. These results indicate that the proton-motive force, in particular the Deltapsi component, plays a role in efficient degradation of substrates by FtsH in its native state. FtsH variants with altered transmembrane regions did not receive proton-motive force stimulation, suggesting that the proton-motive force activates FtsH, directly or indirectly, through the transmembrane region.

Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent metalloprotease. Its cytoplasmic domain contains a zinc-binding motif, H(417)EXXH, whose histidine residues have been shown to be functionally important. Although they are believed to be involved directly in zinc coordination, nothing is known about the third zinc ligand of this protease. Sequence alignment indicates that glutamic acid residues are conserved among the FtsH homologues at positions corresponding to Glu(479) and Glu(585) of E. coli FtsH. We replaced each of them by Gln, Asp, Lys, or Val. Mutations at position 479 compromised the proteolytic functions of FtsH in vivo. In vitro proteolytic activities of the E479Q, E479V, and E479D mutant enzymes were much lower than that of the wild-type protein and were significantly stimulated by a high concentration of zinc ion. These mutant proteins retained the wild-type levels of ATPase activities, and their trypsin susceptibilities as well as CD spectra were essentially indistinguishable from those of the wild-type protein, indicating that the mutations did not cause gross conformational changes in FtsH. They exhibited reduced zinc contents upon purification. From these results, we conclude that Glu(479) is a zinc-coordinating residue.

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β-barrel assembly machinery (BAM) driving integration of β-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein-protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane. [ABSTRACT FROM AUTHOR]

Discusses the cloning, expression, and purification of the SecY protein complex from a thermophilic bacterium, Thermus thermophilus HB8. Use of fluorescence resonance energy transfer analysis; Amino acid sequences of SecY; Purification and oligometric states of detergent-solubilized SecY.