Yoshinori Akiyama

YaeL proteolysis of RseA is controlled by the PDZ domain of YaeL and a Gln-rich region of RseA

σE is an alternative sigma factor involved in a pathway of extracytoplasmic stress responses in Escherichia coli. Under normal growth conditions, σE activity is down‐regulated by the membrane‐bound anti‐σE protein, RseA. Extracytoplasmic stress signals induce degradation of RseA by two successive proteolytic events: DegS‐catalyzed first cleavage at a periplasmic site followed by YaeL‐mediated second proteolysis at an intramembrane region. Normally, the second reaction (site‐2 proteolysis) only occurs after the first cleavage (site‐1 cleavage). Here, we show that YaeL variants with the periplasmic PDZ domain deleted or mutated allows unregulated cleavage of RseA and consequent σE activation. It was also found that a glutamine‐rich region in the periplasmic domain of RseA was required for the avoidance of the YaeL‐mediated proteolysis in the absence of site‐1 cleavage. These results indicate that multiple negative elements both in the enzyme (PDZ domain) and in the substrate (glutamine‐rich region) determine the strict dependence of the site‐2 proteolysis on the site‐1 cleavage.

Quality control of cytoplasmic membrane proteins in Escherichia coli.

In Escherichia coli, like in any organism, the cytoplasmic (inner or plasma) membrane proteins play essential roles in transport of small and macro-molecules as well as in transmission of environmental signals across the membrane. Their quality control is critically important for growth and survival of the cell. However, our knowledge about the players and mechanisms of the system is still limited. This review focuses on proteolytic quality control of membrane proteins, in which two membrane-integrated proteases, FtsH and HtpX, with different modes of action, play central roles. The prohibitin family membrane protein complexes (HflKC and QmcA) contribute to the quality control system as a regulatory factor of FtsH and also as a possible membrane-chaperone. Failure of the quality control system to function normally leads to accumulation of malfolded cytoplasmic membrane proteins, which in turn activate the stress response pathways previously believed to be specialized for sensing protein abnormalities outside the cytoplasmic membrane. In fact, many of the cytoplasmic membrane quality control factors are stress induced. Further characterization of them as well as of the stress-sensing mechanisms would prove useful to obtain an integrated picture of the membrane protein quality control system.

Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease.

Rhomboids are a family of serine proteases belonging to intramembrane cleaving proteases, which are supposed to catalyse proteolysis of a substrate protein within the membrane. It remains unclear whether substrates of the rhomboid proteases have a common sequence feature that allows specific cleavage by rhomboids. We showed previously that GlpG, the Escherichia coli rhomboid, can cleave a type I model membrane protein Bla‐LY2‐MBP having the second transmembrane region of lactose permease (LY2) at the extramembrane region in vivo and in vitro, and that determinants for proteolysis reside within the LY2 sequence. Here we characterized sequence features in LY2 that allow efficient cleavage by GlpG and identified two elements, a hydrophilic region encompassing the cleavage site and helix‐destabilizing residues in the downstream hydrophobic region. Importance of the positioning of helix‐destabilizers relative to the cleavage site was suggested. These two elements appear to co‐operatively promote proteolysis of substrates by GlpG. Finally, random mutagenesis of the cleavage site residues in combination with in vivo screening revealed that GlpG prefers residues with a small side chain and a negative charge at the P1 and P1′ sites respectively.

SecY alterations that impair membrane protein folding and generate a membrane stress

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/σE stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle–targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria

A signal peptide (SP) is cleaved off from presecretory proteins by signal peptidase during or immediately after insertion into the membrane. In metazoan cells, the cleaved SP then receives proteolysis by signal peptide peptidase, an intramembrane-cleaving protease (I-CLiP). However, bacteria lack any signal peptide peptidase member I-CLiP, and little is known about the metabolic fate of bacterial SPs. Here we show that Escherichia coli RseP, an site-2 protease (S2P) family I-CLiP, introduces a cleavage into SPs after their signal peptidase-mediated liberation from preproteins. A Bacillus subtilis S2P protease, RasP, is also shown to be involved in SP cleavage. These results uncover a physiological role of bacterial S2P proteases and update the basic knowledge about the fate of signal peptides in bacterial cells.

Recruitment of a species-specific translational arrest module to monitor different cellular processes

Nascent chain-mediated translation arrest serves as a mechanism of gene regulation. A class of regulatory nascent polypeptides undergoes elongation arrest in manners controlled by the dynamic behavior of the growing chain; Escherichia coli SecM monitors the Sec protein export pathway and Bacillus subtilis MifM monitors the YidC membrane protein integration/folding pathway. We show that MifM and SecM interact with the ribosome in a species-specific manner to stall only the ribosome from the homologous species. Despite this specificity, MifM is not exclusively designed to monitor membrane protein integration because it can be converted into a secretion monitor by replacing the N-terminal transmembrane sequence with a secretion signal sequence. These results show that a regulatory nascent chain is composed of two modular elements, one devoted to elongation arrest and another devoted to subcellular targeting, and they imply that physical pulling force generated by the latter triggers release of the arrest executed by the former. The combinatorial nature may assure common occurrence of nascent chain-mediated regulation.

Nascentome analysis uncovers futile protein synthesis in escherichia coli

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.

Substrate Recognition and Binding by RseP, an Escherichia coli Intramembrane Protease

Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-σE protein RseA as an essential step in transmembrane signal transduction in the σE extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.

Protease homolog BepA (YfgC) promotes assembly and degradation of β-barrel membrane proteins in Escherichia coli

Significance Outer membrane proteins (OMPs) are involved in important cellular activities in Gram-negative bacteria. Although the bepA (formerly yfgC) gene encoding a putative metalloprotease has been implicated in quality control of OMPs, its specific function remains unclear. This study reveals that BepA promotes assembly of LptD, an OMP involved in the transport of lipopolysaccharides, which undergoes intramolecular disulfide rearrangement during its biogenesis. BepA also promotes degradation of incorrectly folded LptD. BamA, another OMP involved in OMP assembly, is also degraded in a BepA-dependent manner in the absence of periplasmic chaperone SurA. BepA thus controls the quality of OMPs by promoting either the biogenesis or elimination of OMPs, depending on their folding state. Gram-negative bacteria are equipped with quality-control systems for the outer membrane (OM) that sense and cope with defective biogenesis of its components. Accumulation of misfolded outer membrane proteins (OMPs) in Escherichia coli leads to activation of σE, an essential alternative σ factor that up-regulates transcription of multiple genes required to preserve OM structure and function. Disruption of bepA (formerly yfgC), a σE-regulated gene encoding a putative periplasmic metalloprotease, sensitizes cells to multiple drugs, suggesting that it may be involved in maintaining OM integrity. However, the specific function of BepA remains unclear. Here, we show that BepA enhances biogenesis of LptD, an essential OMP involved in OM transport and assembly of lipopolysaccharide, by promoting rearrangement of intramolecular disulfide bonds of LptD. In addition, BepA possesses protease activity and is responsible for the degradation of incorrectly folded LptD. In the absence of periplasmic chaperone SurA, BepA also promotes degradation of BamA, the central OMP subunit of the β-barrel assembly machinery (BAM) complex. Interestingly, defective oxidative folding of LptD caused by bepA disruption was partially suppressed by expression of protease-active site mutants of BepA, suggesting that BepA functions independently of its protease activity. We also show that BepA has genetic and physical interaction with components of the BAM complex. These findings raised the possibility that BepA maintains the integrity of OM both by promoting assembly of OMPs and by proteolytically eliminating OMPs when their correct assembly was compromised.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

Significance Bacteria living in seawater must cope with low-sodium environments that they may encounter. Here we show an unexpected finding that remodeling of the Sec protein export machinery plays a pivotal role in this adaptation. Vibrio alginolyticus possesses alternative SecDF1 and SecDF2 homologs that use the transmembrane gradient of Na+ and that of H+, respectively, to enhance protein export by cooperating with the SecYEG translocon. The synthesis of SecDF2 is induced in low-Na+ environments, and this induction is essential for the bacterium to survive low salinity. Remarkably, the Vibrio species use a nascent polypeptide, VemP, to monitor the functional state of the Sec pathway and to up-regulate translation of SecDF2 when activity of the SecDF1-containing Sec machinery declines. 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.