Protein translocation across the peroxisomal membrane

Peroxisomal matrix proteins are synthesized on free cytosolic ribosomes and posttranslationally imported into the organelle. Translocation of these newly synthesized proteins across the peroxisomal membrane requires the concerted action of many different proteins, the majority of which were already identified. However, not much is known regarding the mechanism, of protein translocation across this membrane system. Here, we discuss recent mechanistic and structural data. These results point to a model in which proteins en route to the peroxisomal matrix are translocated across the organelle membrane by their own receptor in a process that occurs, through a large membrane protein assembly.     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.     

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.     

Construction of an Expression System for Aqualysin I in Escherichia coli That Gives a Markedly Improved Yield of the Enzyme Protein

An expression system for aqualysin I from Thermus aquaticus YT-1, a thermophilic serine protease belonging to the proteinase K family, in Escherichia coli is available, but the efficiency of production has been rather low for detailed analysis of the product. We developed a maltose biding protein (MBP)-fused proaqualysin I expression plasmid (pMAQ-c2Δ) in which MBP is attached to the N-terminus of proaqualysin I. MBP appeared effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria were grown at 30 °C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor was converted efficiently to mature aqualysin I by heat treatment at 70 °C, enabling us to obtain 40 times more aqualysin I than is available using expression systems such as pAQNΔC105. By analyzing the product of the pMAQ-c2Δ-derived inactive mutant expression vector, pMAQ-S222A, it was confirmed that aqualysin I was initially expressed as a whole fusion protein and then processed autocatalytically.     

Substrate Specificity of Aqualysin I Altered by an Organic Solvent,DMSO

Aqualysin I is the alkaline serine protease isolated from an extreme thermophile, Thermus aquaticus YT-1. We have analyzed the kinetic properties of aqualysin I, using thirty-one kinds of chromogenic succinyl-tripeptide p-nitroanilides as substrates in the presence of 10% dimethylsulfoxide (DMSO). Aqualysin I hydrolyzed many peptides in a DMSO-containing mixture, however the substrate specificity was different from that in the absence of DMSO. The K_m for each peptide was raised by the addition of 10% DMSO. Also, the P3- as well as P2-specificity of aqualysin I was altered. These results suggested that the side chains of the P2 and P3 residues are exposed to the solvent, and the hydrophobic interactions between the side chain of the substrate and the solvent may take a part in the substrate recognition of the enzyme.     

SecA-dependence of the translocation of a large periplasmic loop in the Escherichia coli MalF inner membrane protein is a function of sequence context

We have analysed the translocation of a large periplasmic loop in the Escherichia coli MalF Inner membrane protein when placed in different sequence contexts and under conditions when the function of the SecA protein is Inhibited. The results show that the degree of SecA-dependence varies with sequence context: while translocation of the large loop In its normal context Is only minimally affected by SecA Inhibition, translocation is much more sensitive to SecA inhibition when the loop is placed in the context of other inner membrane proteins. Conversely, when the large MalF loop is replaced by segments from other proteins, translocation of those segments is again very sensitive to SecA inhibition. Thus, SecA-dependence is not an all-or-none phenomenon and Is not only a simple function of, e.g. the length of a translocated segment or the hydrophobicity of the flanking transmembrane segments.     

SecA protein: Autoregulated initiator of secretory precursor protein translocation across theE. coli plasma membrane

Several classes ofsecA mutants have been isolated which reveal the essential role of this gene product forE. coli cell envelope protein secretion. SecA-dependent,in vitro protein translocation systems have been utilized to show that SecA is an essential, plasma membrane-associated, protein translocation factor, and that SecA's ATPase activity appears to play an essential but as yet undefined role in this process. Cell fractionation studies suggested that SecA protein is in a dynamic state within the cell, occurring in soluble, peripheral, and integral membraneous states. These data have been used to argue that SecA is likely to promote the initial insertion of secretory precursor proteins into the plasma membrane in a manner dependent on ATP hydrolysis. The protein secretion capability of the cell has been shown to translationally regulatesecA expression with SecA protein serving as an autogenous repressor, although the exact mechanism and purpose of this regulation need to be defined further.     

Molecular chaperones and protein translocation across the Escherichia coli inner membrane

Proteins that are able to translocate across biological membranes assume a loosely folded structure. In this review it is suggested that the loosely folded structure, referred to here as the 'pre-folded conformation', is a particular structure that interacts favourably with components of the export apparatus. Two soluble factors, SecB and GroEL, have been implicated in maintenance of the pre-folded conformation and have been termed 'molecular chaperones'. Results suggest that SecB may be a chaperone that is specialized for binding to exported protein precursors, while GroEL may be a general folding modulator that binds to many intracellular proteins.     

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.     

Extracellular Production of a Serratia marcescens Serine Protease in Escherichia coli

The Serratia marcescens serine protease (SSP) is one of the extracellular enzymes secreted from this Gram-negative bacterium. When the ssp gene, which encodes a SSP precursor (preproSSP) composed of a typical NH₂-terminal signal peptide, a mature enzyme domain, and a large COOH-terminal pro-region, is expressed in Escherichia coli, the mature protease is excreted through the outer membrane into the medium. The COOH-terminal pro-region, which is integrated into the outer membrane, provides the essential function for the export of the mature protein across the outer membrane. This is a very simple pathway, in contrast to the general secretory pathway exemplified by the secretion of a pullulanase from Klebsiella oxytoca, in which many separately encoded accessory proteins are required for the transport through the outer membrane. Moreover, the NH₂-terminal region of 71 amino acid residues of the COOH-terminal pro-sequence plays an essential role, as an “intramolecular chaperone,” in the folding of the mature enzyme in the medium. In addition to ssp, the S. marcescens strain contains two ssp homologues encoding proteins similar to SSP in amino acid sequence and size, but with no protease activity. Characterization of the homologue proteins and chimeric proteins between the homologues and SSP, all of which are produced in E. coli, has shown that they are membrane proteins that are localized in the outer membrane in the same manner as for SSP. By use of the COOH-terminal domain of SSP, pseudoazurin was exported to the cell surface of E. coli, which proves the usefulness of the SSP secretory system in the export of foreign proteins across the outer membrane.     

A non-covalent NH2-terminal pro-region aids the production of active aqualysin I (a thermophilic protease) without the COOH-terminal pro-sequence in Escherichia coli

The precursor of aqualysin I, an extracellular protease produced by Thermus aquaticus, consists of four domains: an N-terminal signal peptide, an N-terminal pro-sequence, the protease domain and a C-terminal pro-sequence. In an Escherichia coli expression system, mature and active aqualysin I is formed by treatment at 65 degrees C and the N-pro-sequence is required for its production. Complete deletion of the C-pro-sequence did not affect the production of active aqualysin I, indicating that the C-pro-sequence is not essential. A non-covalent N-pro-region was separately synthesized from the protease domain with or without the C-pro-sequence. In this system, mature and active aqualysin I was detected only when the C-pro-sequence was deleted.     

Substrate-specific function of the translocon-associated protein complex during translocation across the ER membrane

Although the transport of model proteins across the mammalian ER can be reconstituted with purified Sec61p complex, TRAM, and signal recognition particle receptor, some substrates, such as the prion protein (PrP), are inefficiently or improperly translocated using only these components. Here, we purify a factor needed for proper translocation of PrP and identify it as the translocon-associated protein (TRAP) complex. Surprisingly, TRAP also stimulates vectorial transport of many, but not all, other substrates in a manner influenced by their signal sequences. Comparative analyses of several natural signal sequences suggest that a dependence on TRAP for translocation is not due to any single physical parameter, such as hydrophobicity of the signal sequence. Instead, a functional property of the signal, efficiency of its post-targeting role in initiating substrate translocation, correlates inversely with TRAP dependence. Thus, maximal translocation independent of TRAP can only be achieved with a signal sequence, such as the one from prolactin, whose strong interaction with the translocon mediates translocon gating shortly after targeting. These results identify the TRAP complex as a functional component of the translocon and demonstrate that it acts in a substrate-specific manner to facilitate the initiation of protein translocation.     

Role of amino-terminal positive charge on signal peptide in staphylokinase export across the cytoplasmic membrane of Escherichia coli

Staphylokinase mutants having amino acid substitutions within the amino-terminal charged segment of the signal peptide have been produced by in vitro oligonucleotide-directed mutagenesis. When the processing of the gene products was analyzed in Escherichia coli cells, the rate of processing of the mutant staphylokinase precursor decreased as the net charge became more negative. A net positive charge, but not specific amino acid residues, was required on the amino-terminal segment for efficient processing. Staphylokinase precursor having a net negative charge accumulated in the cytoplasm, tending to bind to the cytoplasmic membrane as determined by subcellular fractionation and immunoelectron microscopy. Although a mutant carrying an amino acid substitution in the hydrophobic segment and wild-type staphylokinases had an interfering effect on the processing of other normal secreted proteins, this effect was lost when they also contained charge-altering substitutions in the amino-terminal region. From these results, we concluded that a positive charge on the amino-terminal segment of the staphylokinase signal peptide is required for entrance into the protein export process.     

Peculiar properties of DsbA in its export across the Escherichia coli cytoplasmic membrane

Export of DsbA, a protein disulfide bond-introducing enzyme, across the Escherichia coli cytoplasmic membrane was studied with special reference to the effects of various mutations affecting translocation factors. It was noted that both the internalized precursor retaining the signal peptide and the periplasmic mature product fold rapidly into a protease-resistant structure and they exhibited anomalies in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in that the former migrated faster than the latter. The precursor, once accumulated, was not exported posttranslationally. DsbA export depended on the SecY translocon, the SecA ATPase, and Ffh (signal recognition particle), but not on SecB. SecY mutations, such as secY39 and secY205, that severely impair translocation of a number of secretory substrates by interfering with SecA actions only insignificantly impaired the DsbA export. In contrast, secY125, affecting a periplasmic domain and impairing a late step of translocation, exerted strong export inhibition of both classes of proteins. These results suggest that DsbA uses not only the signal recognition particle targeting pathway but also a special route of translocation through the translocon, which is hence suggested to actively discriminate pre-proteins.     

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b5 precursor is an exocytoplasmic post-translocational event in Escherichia coli

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b₅ precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.     

Role of the COOH-terminal pro-sequence of aqualysin I (a heat-stable serine protease) in its extracellular secretion by Thermus thermophilus

Aqualysin I is a subtilisin-type serine protease secreted into the medium by Thermus aquaticus YT-1. Thermus thermophilus cells harboring a plasmid for the aqualysin I precursor secreted pro-aqualysin I with the C-terminal pro-sequence into the culture medium, and the precursor was then processed to the mature enzyme during the cultivation. However, the extracellular levels of aqualysin I in T. thermophilus cells harboring plasmids for deletion mutants as to the C-terminal pro-sequence were about 10–20% in comparison with the level of wild-type. Only the mature enzyme could be detected in the medium, while pro-aqualysin I with the C-terminal pro-sequence could not. These results suggest that the C-terminal pro-sequence of aqualysin I plays an important role in the extracellular secretion of aqualysin I.     

Kinetic analysis of the translocation of fluorescent precursor proteins into Escherichia coli membrane vesicles

Protein secretion in Escherichia coli is mediated by translocase, a multi-subunit membrane protein complex with SecA as ATP-driven motor protein and the SecYEG complex as translocation pore. A fluorescent assay was developed to facilitate kinetic studies of protein translocation. Single cysteine mutants of proOmpA were site-specific labeled with fluorescent dyes, and the SecA and ATP-dependent translocation into inner membrane vesicles and SecYEG proteoliposomes was monitored by means of protease accessibility and in gel fluorescent imaging. The translocation of fluorescently labeled proOmpA was largely independent on the position and the size of the fluorescent label (up to a size of 13-16 A). A fluorophore at the +4 position blocked translocation, but inhibition was completely relieved in the PrlA4 mutant. The kinetics of translocation of the fluorescently labeled proOmpA could be directly monitored by means of fluorescence quenching. Inner membrane vesicles containing wild-type SecYEG were found to translocate proOmpA with a turnover of 4.5 molecules proOmpA/SecYEG complex/min and an apparent K(m) of 180 nm, whereas the PrlA4 mutant showed an almost 10-fold increase in turnover rate and a 3-fold increase of the apparent K(m) for proOmpA translocation.