Ken-ichi Nishiyama

Inversion of the Membrane Topology of SecG Coupled with SecA-Dependent Preprotein Translocation

E. coli preprotein translocase comprises SecA and SecY/E/G complex. SecA delivers the preprotein to the putative protein-conducting channel formed by SecY/E by undergoing ATP-driven cycles of membrane insertion and deinsertion. SecG renders the translocase highly efficient. An antibody raised against the C-terminal region of SecG inhibits preprotein translocation into everted membrane vesicles despite the exposure of this region to the inside of membrane vesicles in the absence of preprotein translocation. When preprotein translocation was started with ATP and then blocked by the inhibition of ATP hydrolysis, the C-terminal region was exposed to the outside of membrane vesicles. Another region of SecG showed a change in membrane sidedness upon preprotein translocation, indicating that SecG undergoes topology inversion. This topology inversion was tightly coupled to the SecG function and linked with the insertion-deinsertion cycle of SecA.

Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature.

The Escherichia coli cytoplasmic membrane protein, p12, stimulates the protein translocation activity reconstituted with SecY, SecE and SecA. The gene encoding p12, which is located at 69 min on the E. coli chromosome, was deleted to examine the role of p12 in protein translocation in vivo. The deletion strain exhibited cold‐sensitive growth. Pulse‐chase experiments revealed that precursors of outer membrane protein A, maltose binding protein and beta‐lactamase accumulated at 20 degrees C but not at 37 degrees C. The deletion strain harboring a plasmid which carries the gene encoding p12 under the control of the araBAD promoter was able to grow in the cold when p12 was expressed with the addition of arabinose. Furthermore, the accumulated precursors were rapidly processed to the mature forms upon the expression of p12. Immunoblot analysis revealed the steady‐state accumulation of precursor proteins at 20 degrees C, whereas the accumulation was only marginal at 37 degrees C, indicating that the function of p12 is more critical at 20 degrees C than at 37 degrees C. Finally, proteoliposomes were reconstituted with or without p12 to demonstrate that the stimulation of the activity by p12 increases with a decrease in temperature. From these results, we concluded that p12 is directly involved in protein translocation in E. coli and plays a critical role in the cold. We propose the more systematic name, SecG, for p12.

A novel membrane protein involved in protein translocation across the cytoplasmic membrane of Escherichia coli.

A novel factor, which is a membrane component of the protein translocation machinery of Escherichia coli, was discovered. This factor was found in the trichloracetic acid‐soluble fraction of solubilized cytoplasmic membrane. The factor was purified to homogeneity by ion exchange column chromatographies and found to be a hydrophobic protein with a molecular mass of approximately 12 kDa. The factor caused > 20‐fold stimulation of the protein translocation when it was reconstituted into proteoliposomes together with SecE and SecY. SecE, SecY, SecA and ATP were essential for the factor‐dependent stimulation of the activity. The factor stimulated the translocation of all three precursor proteins examined, including authentic proOmpA. Stimulation of the translocation of proOmpF‐Lpp, a model presecretory protein, was especially remarkable, since no translocation was observed unless proteoliposomes were reconstituted with the factor. Partial amino acid sequence of the purified factor was determined. An antibody raised against a synthetic peptide of this sequence inhibited the protein translocation into everted membrane vesicles, indicating that the factor is playing an important role in protein translocation into membrane vesicles. The partial amino acid sequence was found to coincide with that deduced from the reported DNA sequence of the upstream region of the leuU gene. Cloning and sequencing of the upstream region revealed the presence of a new open reading frame, which encodes a hydrophobic protein of 11.4 kDa. We propose that the factor is a general component of the protein translocation machinery of E. coli.

Structural basis of Sec-independent membrane protein insertion by YidC

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Membrane deinsertion of SecA underlying proton motive force‐dependent stimulation of protein translocation

The proton motive force (PMF) renders protein translocation across the Escherichia coli membrane highly efficient, although the underlying mechanism has not been clarified. The membrane insertion and deinsertion of SecA coupled to ATP binding and hydrolysis, respectively, are thought to drive the translocation. We report here that PMF significantly decreases the level of membrane‐inserted SecA. The prlA4 mutation of SecY, which causes efficient protein translocation in the absence of PMF, was found to reduce the membrane‐inserted SecA irrespective of the presence or absence of PMF. The PMF‐dependent decrease in the membrane‐inserted SecA caused an increase in the amount of SecA released into the extra‐membrane milieu, indicating that PMF deinserts SecA from the membrane. The PMF‐dependent deinsertion reduced the amount of SecA required for maximal translocation activity. Neither ATP hydrolysis nor exchange with external SecA was required for the PMF‐dependent deinsertion of SecA. These results indicate that the SecA deinsertion is a limiting step of protein translocation and is accelerated by PMF, efficient protein translocation thereby being caused in the presence of PMF.

Development of a Minimal Cell-Free Translation System for the Synthesis of Presecretory and Integral Membrane Proteins

By combining translation and membrane integration/translocation systems, we have constructed a novel cell‐free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell‐free system reconstituted from a minimal number of translation factors was supplemented with urea‐washed inverted membrane vesicles (U‐INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U‐INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell‐free system. Thus, our novel cell‐free system provides not only an efficient strategy for the production of membrane‐related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.

Coupled structure change of SecA and SecG revealed by the synthetic lethality of the secAcsR11 and ΔsecG::kan double mutant

An Escherichia coli strain carrying either the secAcsR11 or ΔsecG::kan mutation is unable to grow at low temperature owing to cold‐sensitive protein translocation but grows normally at 37°C. However, introduction of the two mutations into the same cells caused a severe defect in protein translocation and the cells were unable to grow at any temperature examined, indicating that secG is essential for the secAcsR11 mutant. The mutant SecA (csSecA) was found to possess a single amino acid substitution in the precursor‐binding region and was defective in the interaction with the precursor protein. Furthermore, the membrane insertion of SecA and the membrane topology inversion of SecG, both of which took place upon the initiation of protein translocation, were significantly retarded even at 37°C, when csSecA was used instead of the wild‐type SecA. The insertion of the wild‐type SecA was also significantly defective when SecG‐depleted membrane vesicles were used in place of SecG‐containing ones. No insertion of csSecA occurred into SecG‐depleted membrane vesicles. Examination of in vitro protein translocation at 37°C revealed that SecG is essential for csSecA‐dependent protein translocation. We conclude that SecG and SecA undergo a coupled structure change, that is critical for efficient protein translocation.

The periplasmic chaperone PpiD interacts with secretory proteins exiting from the SecYEG translocon.

The Sec translocon of Escherichia coli mediates the export of numerous secretory and membrane proteins. To dissect the passage of an exported protein across the Sec translocon into consecutive steps, we generated in vitro translocation intermediates of a polypeptide chain, which by its N-terminus is anchored in the membrane and by its C-terminus tethered to the ribosome. We find that in this situation, the motor protein SecA propagates translocation of a peptide loop across SecYEG prior to the removal of ribosomes. Upon SecA-driven exit from the translocon, this loop is brought into the immediate vicinity of the membrane-anchored, periplasmic chaperone PpiD. Consistent with a coupling between translocation across the SecYEG translocon and folding by periplasmic chaperones, a lack of PpiD retards the release of a translocating outer membrane protein into the periplasm.

SecG plays a critical role in protein translocation in the absence of the proton motive force as well as at low temperature

SecG is an integral membrane component of E. coli protein translocase. However, a discrepancy exists as to the importance of SecG for protein translocation at 37°C between cells and reconstituted proteoliposomes; protein translocation in ΔsecG cells is defective at 20°C but normal at 37°C, indicating that SecG is dispensable at 37°C, whereas SecG remarkably stimulates protein translocation into reconstituted proteoliposomes at 37°C. In this study, protein translocation into membrane vesicles containing or not containing SecG was examined in the presence and absence of the proton motive force at 37°C and 20°C. We found that the absence of the proton motive force renders protein translocation strongly dependent on SecG even at 37°C. Protein translocation into proteoliposomes in the absence of the proton motive force thus required SecG whereas that in cells, which always generate the proton motive force, did not.

Expression of gpsA encoding biosynthetic sn‐glycerol 3‐phosphate dehydrogenase suppresses both the LB− phenotype of a secB null mutant and the cold‐sensitive phenotype of a secG null mutant

SecB maintains the structures of a subset of precursor proteins competent for translocation across the Escherichia coli cytoplasmic membrane. SecG, a membrane component of the translocation machinery, stimulates protein translocation by undergoing the cycle of membrane topology inversion. Null mutants of secB and secG are unable to form isolated colonies on rich medium and at low temperature respectively. A 3.2 kb DNA fragment carrying the secB–gpsA region on a multicopy plasmid was found to suppress the null mutation of either gene. However, subcloning of the DNA fragment revealed that secB is not involved in the suppression of either mutation. Instead, gpsA located downstream from the secB gene was found to be responsible for the suppression of both mutations. The activity of the gpsA‐encoded sn‐glycerol‐3‐phosphate dehydrogenase, which is involved in phospholipid synthesis, was significantly lower in the secB null mutant than in the wild type, presumably because of a polar effect. Suppression of the secB null mutation required the wild‐type level of GpsA activity. In contrast, overexpression of the enzyme was essential for suppression of the secG null mutation. Moreover, the gpsA‐dependent suppression of the secG null mutation occurred only on rich medium, i.e. not on minimal medium. These results indicate that the SecB function is dispensable even in rich medium, and further demonstrate that overexpression of enzymes involved in phospholipid synthesis partly compensates for the SecG function.