The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides.

A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.     

In vitro translocation of bacterial secretory proteins and energy requirements

The recent establishment ofin vitro assay systems has made biochemical studies on the process of membrane translocation of secretory proteins possible. This review summarizes what we have learned, using thesein vitro systems, concerning the biochemical process of protein translocation, with special reference to energy requirements. Both ATP and the protonmotive force participate in the translocation reaction. The requirement of ATP is obligatory, whereas that of the protonmotive force differs, in terms of its level, with the secretory protein species. The possible roles of ATP and the protonmotive force in protein translocation are discussed with special reference to the function of SecA, an essential component of the secretory machinery. The effect of positive charges, which precede or follow the hydrophobic domain of signal peptides, on translocation is also discussed.     

Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli. Relation to the orientation of membrane proteins

The introduction of positive charges at the amino terminus of the mature domain of secretory proteins resulted in strong inhibition of their translocation across the cytoplasmic membrane of Escherichia coli, both in vitro and in vivo. The model secretory proteins used were OmpF-Lpp chimeric proteins possessing a cleavable or uncleavable signal peptide, beta-lactamase (Bla) and Bla-Lpp chimeric proteins. It is suggested that positively charged residues preceding the hydrophobic domain of the signal peptide have a positive effect, and ones following the hydrophobic domain, a negative effect on the translocation. These findings are discussed in relation to the orientation of membrane proteins, of which positive charges are predominant on the cytoplasmic surface.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

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.     

A proline residue near the amino terminus of the mature domain of secretory proteins lowers the level of the proton motive force required for translocation.

A large variety of proOmpF-Lpps, hybrid secretory proteins composed of the signal region of proOmpF and the mature part of the major lipoprotein, either possessing or not possessing a proline residue near the amino terminus of their mature domains, were constructed at a DNA level, and the rates of their in vitro translocation were determined in the presence and absence of the proton motive force (delta muH+). A proline residue at the signal peptide cleavage site (position +1) blocked the cleavage reaction but not the translocation reaction. All the proOmpF-Lpps examined exhibited approximately the same translocation rate in the presence of delta muH+ irrespective of the presence or absence of a proline residue near the amino terminus. In the absence of the delta muH+, which was achieved by either depletion of the respiratory substrate or the use of urea-treated membrane vesicles permeable to protons, proOmpF-Lpps possessing a proline residue near the amino terminus of the mature domain were translocated whereas those possessing no proline residue in this region were not translocated at all or only very weakly. The position of the proline residue was then moved stepwise away from the amino terminus of the mature domain. The further the position was moved away, the slower was the rate of translocation in the absence of delta muH+. The removal of the proline residue at position +2 of the mature domain of proOmpA also made the delta mu(H+)-independent translocation appreciably slower. It is suggested that the conformational flexibility endowed by the proline residue on the junction region between the signal peptide and the mature domain allows the translocation in the absence of delta muH+ and that this junction region must take on a particular conformation for initiation of the translocation reaction.     

Effects of total hydrophobicity and length of the hydrophobic domain of a signal peptide on in vitro translocation efficiency.

The hydrophobic domain of the signal peptide of OmpF-Lpp, a model secretory protein, was systematically engineered so as to be composed of different lengths of polyleucine residues or polymers with alternate leucine and alanine residues, and the effects of the length and nature of the hydrophobic stretch on the rate of in vitro translocation were studied using everted membrane vesicles of Escherichia coli. The translocation reaction exhibited high substrate specificity as to the number of hydrophobic residues. The results suggest that the hydrophobic domain is recognized specifically by a component(s) of the secretory machinery rather than nonspecifically by the hydrophobic region of the membrane. The in vitro translocation thus demonstrated required SecA and ATP and was markedly enhanced upon imposition of the proton motive force, as in the case of secretory proteins possessing a natural signal peptide. The highest translocation rate was obtained with the octamer in the case of polyleucine-containing signal peptides, whereas it was the decamer in the case of ones containing both leucine and alanine. These results suggest that the total hydrophobicity of the hydrophobic region of the signal peptides is an important determinant of the substrate specificity.     

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.     

A high concentration of SecA allows proton motive force-independent translocation of a model secretory protein into Escherichia coli membrane vesicles

The in vitro translocation of OmpF-Lpp, a model secretory protein, into inverted membrane vesicles of Escherichia coli obligatorily requires the proton motive force (delta mu H+) in the conventional assay system (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The translocation, however, took place efficiently, even in the absence of delta mu H+, when the system was supplemented with additional SecA. With the stripped membrane vesicles, which are permeable to protons, or in the absence of NADH, the supplementation of SecA remarkably stimulated the translocation activity. The further addition of NADH did not significantly enhance the translocation activity under the SecA-enriched conditions. OmpF-Lpp thus translocated could be recovered from the vesicular lumen by sonication, indicating that complete translocation occurred in the absence of delta mu H+. It is suggested that delta mu H+ is required for high affinity interaction of SecA with the presumed secretory machinery in the cytoplasmic membrane and that a high concentration of SecA modulates the delta mu H+ requirement.     

Demonstration of post-translational secretion of human placental lactogen by a mammalian in vitro translation system

This study demonstrates the post-translational translocation across the rough endoplasmic reticular membrane of a mammalian secretory protein, human preplacental lactogen. In the rabbit reticulocyte lysate, human preplacental lactogen biosynthesis is arrested by addition of cycloheximide prior to supplementation with dog pancreatic microsomal membranes, which have previously been shown to translocate and process nascent secretory proteins in a cotranslational manner. Twenty-five percent of the precursor protein is consistently converted to its mature form under these post-translational conditions. The resulting mature hormone is resistant to proteolytic degradation by added proteases, thus indicating that it is translocated across the microsomal membrane and sequestered within the lumenal space of the microsomal vesicles. Approximately one-half of the precursor protein synthesized is associated with the ribosomes. Only the ribosome-associated fraction is secreted in this in vitro system, suggesting that the process of post-translational secretion requires ribosomes for protein interaction with the elements of a subcellular secretory apparatus.     

Alteration of N-terminal residues of mature human lysozyme affects its secretion in yeast and translocation into canine microsomal vesicles

Signal sequences play a central role in the initial membrane translocation of secretory proteins. Their functions depend on factors such as hydrophobicity and conformation of the signal sequences themselves. However, some characteristics of mature proteins, especially those of the N-terminal region, might also affect the function of the signal sequences. To examine this possibility, several mutants of human lysozyme modified in the N-terminal region of the mature protein were constructed, and their secretion in yeast as well as in vitro translocation into canine pancreatic microsomes were analyzed using an idealized signal sequence L8 (MR(L)8PLAALG). Our results show the following. (1) Change in the charge at the N-terminal residue of the mature protein does not affect secretion drastically. (2) Substitution of a proline residue at the N terminus prevents cleavage of the signal sequence, although translocation itself is not impaired. (3) Excessive positive charges in the N-terminal region delay translocation of the precursor protein across the membrane. (4) Polar and negatively charged residues introduced into the N-terminal region affect the secretion of the mature protein by preventing its correct folding.     

Sec72p contributes to the selective recognition of signal peptides by the secretory polypeptide translocation complex

SEC72 encodes the 23-kD subunit of the Sec63p complex, an integral ER membrane protein complex that is required for translocation of presecretory proteins into the ER of Saccharomyces cerevisiae. DNA sequence analysis of SEC72 predicts a 21.6-kD protein with neither a signal peptide nor any transmembrane domains. Antibodies directed against a carboxyl-terminal peptide of Sec72p were used to confirm the membrane location of the protein. SEC72 is not essential for yeast cell growth, although an sec72 null mutant accumulates a subset of secretory precursors in vivo. Experiments using signal peptide chimeric proteins demonstrate that the sec72 translocation defect is associated with the signal peptide rather than with the mature region of the secretory precursor.     

Position-dependent effects of polylysine on Sec protein transport

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.     

Structural and functional characterization of Sec66p, a new subunit of the polypeptide translocation apparatus in the yeast endoplasmic reticulum.

SEC66 encodes the 31.5-kDa glycoprotein of the Sec63p complex, an integral endoplasmic reticulum membrane protein complex required for translocation of presecretory proteins in Saccharomyces cerevisiae. DNA sequence analysis of SEC66 predicts a 23-kDa protein with no obvious NH2-terminal signal sequence but with one domain of sufficient length and hydrophobicity to span a lipid bilayer. Antibodies directed against a recombinant form of Sec66p were used to confirm the membrane location of Sec66p and that Sec66p is a glycoprotein of 31.5 kDa. A null mutation in SEC66 renders yeast cells temperature sensitive for growth. sec66 cells accumulate some secretory precursors at a permissive temperature and a variety of precursors at the restrictive temperature. sec66 cells show defects in Sec63p complex formation. Because sec66 cells affect the translocation of some, but not all secretory precursor polypeptides, the role of Sec66p may be to interact with the signal peptide of presecretory proteins.     

In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force

The proton motive force (delta mu H+) plays an important role, although it is not absolutely essential, in the in vitro translocation of secretory proteins, such as OmpA, across the cytoplasmic membrane of Escherichia coli (Yamada, H., Tokuda, H., and Mizushima, S. (1989) J. Biol. Chem. 264, 1723-1728). The transient accumulation in membrane vesicles of a possible translocation intermediate of OmpA was observed in the absence of delta mu H+. The intermediate was detected on a polyacrylamide gel as a proteinase K-resistant band corresponding to a molecular weight of 26,000. The intermediate did not possess the signal peptide. The appearance of this band was inhibited in the absence of ATP or the presence of adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) and enhanced upon the addition of SecA. Upon the addition of NADH that energizes the membrane, the intermediate was converted to the translocated form of OmpA, even in the presence of AMP-PNP. These results suggest different requirements of ATP and delta mu H+ for the early and late stages of the translocation reaction. The SecA requirement for the early stage of the translocation has also been suggested. In addition to this band, two other bands were observed at higher positions on the gel, when the translocation reaction was performed in the absence of delta mu H+. Although these two bands also represented the mature form of OmpA, which was partly protected from the proteinase K treatment by the membrane vesicles, the accumulation was not transient. These bands did not appear when the translocation reaction was performed in the presence of dithiothreitol. Together with other evidence, the above observations suggest that OmpA, which has an intramolecular disulfide bridge, cannot undergo the translocation unless delta mu H+ is imposed.     

The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism.

The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N-terminal tail. For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain. When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential. In contrast, when a positively charged residue is present within the N-terminal tail of leader peptidase, the potential impedes translocation of the tail domain. However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential. Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation. The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.     

A chemically cross-linked nonlinear proOmpA molecule can be translocated into everted membrane vesicles of Escherichia coli in the presence of the proton motive force.

The chemical cross-linking between the two cysteine residues at positions + 290 and + 302 of proOmpA was performed with N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine. In the absence of the proton motive force (delta muH+), the cross-linked proOmpA was only partially translocated into everted membrane vesicles, leading to accumulation of translocation intermediates. In the presence of delta mu H+, the cross-linked proOmpA was completely translocated. The translocated OmpA still possessed the cross-linked loop composed of 13 amino acid residues and the cross-linker. It is concluded that polypeptide chains need not be necessarily linear and fully expanded to be translocated.     

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.     

Determination of a region in SecA that interacts with presecretory proteins in Escherichia coli.

SecA interacts with presecretory proteins through recognition of the positive charge at the amino terminus of the signal peptide (Akita, M., Sasaki, S., Matsuyama, S., and Mizushima, S. (1989) J. Biol. Chem. 265, 8164-8169). A large variety of amino-terminal and carboxyl-terminal fragments of SecA were prepared in 6 M guanidine hydrochloride. SecA analogues were then reconstituted from them and examined for their ability to cross-link with [35S]proOmpF-Lpp, a presecretory protein, in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The reconstituted SecA analogues were active in the cross-linking with proOmpF-Lpp when the SecA fragments used were large enough to structurally complement each other. The cross-linking was signal peptide-dependent and suppressed in the presence of other presecretory proteins. The cross-linking was enhanced in the presence of ATP. The SecA fragments that cross-linked with proOmpF-Lpp were then analyzed on sodium dodecyl sulfate-polyacrylamide gels. The cross-linking preferentially took place on fragments possessing the amino terminus of SecA. Weak cross-linking was also observed with carboxyl-terminal fragments when they were large enough. The smallest amino-terminal and carboxyl-terminal fragments with which the cross-linking was observed were 39 and 72 kDa, respectively. From these results, the region responsible for the cross-linking with presecretory proteins was deduced to be located between amino acid residues 267 and 340 from the amino terminus of SecA. These results are discussed in relation to the structure and function of SecA.