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PubMed2002

Juxtaposition of signal-peptide charge and core region hydrophobicity is critical for functional signal peptides.

In Escherichia coli, exported proteins are synthesized as precursors containing an amino-terminal signal peptide which directs transport through the translocase to the proper destination. We have constructed a series of signal peptide mutants, incorporating linker sequences of varying lengths between the amino-terminal charge and core region hydrophobicity, to examine the requirement for the juxtaposition of these two structural features in promoting protein transport. In vivo and in vitro analyses indicated that high transport efficiency via signal peptides with core regions of marginal hydrophobicity absolutely requires the proximity of sufficient charge.

Adenosine Triphosphatases↗

PubMed2006

Evaluation of TatABC overproduction on Tat- and Sec-dependent protein secretion in Streptomyces lividans.

The majority of bacterial proteins are exported across the cytoplasmic membrane via the Sec pathway, but also the more recently discovered twin-arginine translocation (Tat) route seems to play an important role for protein secretion in Streptomyces lividans in whose genome tatA, tatB and tatC have been identified. In the present work we showed that simultaneous overproduction of TatABC improved the Tat-dependent secretion capacity as could be concluded from the increased amount of secreted xylanase C, an exclusive Tat-dependent substrate. This result demonstrates that next to the availability of energy to drive secretion, also the number of translocases can be rate-limiting for Tat-dependent secretion. On the other hand, tatABC overexpression was found to diminish secretion of the Sec-dependent proteins xylanase B and subtilisin inhibitor in S. lividans. These results reveal cross-talk between both pathways in S. lividans.

Adenosine Triphosphatases↗

PubMed2004

Secretory and extracellular production of recombinant proteins using Escherichia coli.

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. However, there are often problems in recovering substantial yields of correctly folded proteins. One approach to solve these problems is to have recombinant proteins secreted into the periplasmic space or culture medium. The secretory production of recombinant proteins has several advantages, such as simplicity of purification, avoidance of protease attack and N-terminal Met extension, and a better chance of correct protein folding. In addition to the well-established Sec system, the twin-arginine translocation (TAT) system has recently been employed for the efficient secretion of folded proteins. Various strategies for the extracellular production of recombinant proteins have also been developed. For the secretory production of complex proteins, periplasmic chaperones and protease can be manipulated to improve the yields of secreted proteins. This review discusses recent advances in secretory and extracellular production of recombinant proteins using E. coli.

Adenosine Triphosphatases↗

PubMed2005

The yeast split-ubiquitin system to study chloroplast membrane protein interactions.

Each photosynthetic complex within the thylakoid membrane consists of several different subunits. During formation of these complexes, numerous regulatory factors are required for the coordinated transport and assembly of the subunits. Interactions between transport/assembly factors and their specific polypeptides occur in a membraneous environment and are usually transient and short-lived. Thus, a detailed analysis of the underlying molecular mechanisms by biochemical techniques is often difficult to perform. Here, we report on the suitability of a genetic system, i.e. the yeast split-ubiquitin system, to investigate protein-protein interactions of thylakoid membrane proteins. The data confirm the previously established binding of the cpSec-translocase subunits, cpSecY and cpSecE, and the interaction of the cpSec-translocase from Arabidopsis thaliana with Alb3, a factor required for the insertion of the light-harvesting chlorophyll-binding proteins into the thylakoid membrane. In addition, the proposed interaction between D1, the reaction center protein of photosystem II and the soluble periplasmic PratA factor from Synechocystis sp. PCC 6803 was verified. A more comprehensive analysis of Alb3-interacting proteins revealed that Alb3 is able to form dimers or oligomers. Interestingly, Alb3 was also shown to bind to the PSII proteins D1, D2 and CP43, to the PSI reaction center protein PSI-A and the ATP synthase subunit CF(0)III, suggesting an important role of Alb3 in the assembly of photosynthetic thylakoid membrane complexes.

Arabidopsis↗

PubMed2003

Cloning of the Pichia anomala SEC61 gene and its expression in a Saccharomyces cerevisiae sec61 mutant.

In several organisms, including Saccharomyces cerevisiae and other yeast species, the product encoded by the SEC61 gene is considered to be the core element of the translocation apparatus within the endoplasmic reticulum membrane through which translocation of secretory and membrane proteins occurs. In this study, we have cloned and characterized the homolog of the SEC61 gene from the yeast Pichia anomala. The cloned gene includes an ORF, interrupted after the first ten nucleotides by an intron of 131 bp, encoding a 479-amino acid putative polypeptide exhibiting homology to the products encoded by different eukaryotic SEC61 genes, particularly to those from other yeast species. We show that the P. anomala SEC61 gene is correctly processed (intron splicing) when expressed in S. cerevisiae and that it is able to complement the thermosensitive phenotype associated with a mutation in the S. cerevisiae SEC61 gene.

Cloning, Molecular↗

PubMed1999

Regulation of the Escherichia coli secA gene is mediated by two distinct RNA structural conformations.

Expression from the secA gene, encoding a key component of the general secretory pathway of Escherichia coli, is influenced by the secretion status of the cell, autogenous translational repression, and translational coupling to the upstream gene, X. SecA binds to its mRNA in a region overlapping its ribosome binding site, thus competing with ribosomes that would initiate secA translation. Mapping of the geneX-secA mRNA secondary structure has demonstrated that the RNA can adopt two distinct conformations in solution. The first conformation arises from the base-pairing of the secA Shine-Dalgarno (SD) sequence with the geneX terminus. The second conformation, in which the secA SD sequence is no longer paired with the geneX terminus, contains a GC-rich stem upstream of the secA SD sequence. The presence of this GC-rich stem is supported by structure mapping of a mutant RNA containing a deletion in the geneX terminus. The former structure appears to be involved in translational coupling by directly linking the geneX and secA sequences, where geneX translation activates secA translational initiation through the unpairing and unmasking of the secA SD sequence. As indicated by SecA-RNA binding assays, the latter structure is probably involved in SecA binding and translational repression of the secA gene. The stabilizing effect of magnesium ions toward occlusion of the secA SD sequence supports the presence of RNA tertiary structure in this regulatory domain.

Adenosine Triphosphatases↗

PubMed1998

Phylogenetic analysis of plastid origins based on secA sequences.

We have generated secA sequence data from a number of photosynthetic prokaryotes and carried out a phylogenetic analysis using secA sequences from prokaryotes, green plants, and red and brown algae. We have studied the substitution patterns that give rise to the apparent phylogenetic structure. We show that the high AT content of the plastid sequences significantly affects the amino-acid composition. We also show that most of the apparent evidence for an edge separating red and brown plastids from green plants within the phylogenetic tree is due to differences in nucleotide composition. The remaining apparent evidence is likely to be due, at least in part, to differences in the distribution of sites free to vary. We discuss the implications of this study for hypotheses of plastid origins.

Adenosine Triphosphatases↗

PubMed2002

The Trichoderma atroviride seb1 (stress response element binding) gene encodes an AGGGG-binding protein which is involved in the response to high osmolarity stress.

The chitinase genes of Trichoderma spp. (ech42, chit33, nag1) contain one or more copies of a pentanucleotide element (5'-AGGGG-3') in their 5'-noncoding regions. In Saccharomyces cerevisiae, this motif is recognized and bound by the stress response regulator proteins Msn2p/Msn4p. To test whether this motif in the chitinase promoters is bound by a Trichoderma Msn2/4p homolog, we have cloned a gene (seb1) from T. atroviride which encodes a C2H2 zinc-finger protein that is 62 (64)% identical to S. cerevisiae Msn2p (Msn4p) in the zinc-finger region, and almost identical to the G-box binding protein from Haematonectria haematococca and to polypeptides encoded by uncharacterized ORFs from Neurospora crassa and Aspergillus nidulans. Its zinc-finger domain specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band-shift assays. However, a cDNA clone of seb1, when overexpressed in S. cerevisiae, was unable to complement a Delta msn2/4 mutant of S. cerevisiae. Levels of seb1 mRNA increased under conditions of osmotic stress (sorbitol, NaCl) but not under other stress conditions (cadmium sulfate, pH, membrane perturbance). A T. atroviride Delta seb1 strain, produced by transformation with a seb1 copy disrupted by insertion of the A. nidulans amdS gene, showed strongly reduced growth on solid medium, but grew normally in liquid medium. In liquid medium, growth of the disruption strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than was that of the wild-type strain. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and the disruption strain. EMSA analyses with cell-free extracts obtained from the seb1 disruption strain showed the presence of proteins that could bind to the AGGGG-element in nag1 and ech42. We therefore conclude that seb1 encodes a protein that is involved in the osmotic stress response, but not in chitinase gene expression, in T. atroviride.

Chitinases↗

PubMed2000

SecG is an auxiliary component of the protein export apparatus of Escherichia coli.

SecY, SecE and SecG form the membrane-embedded core complex of the Escherichia coli protein export apparatus. These three proteins co-purify and can be co-immunoprecipitated, demonstrating that they are closely associated. While SecE and SecY are generally accepted as essential components of translocase, the role of SecG is more ambiguous. It is commonly believed that deletion of secG causes a cold-sensitive phenotype and a severe defect in export, even though some reports have indicated otherwise. However, we demonstrate that deletion of secG does not produce a cold-sensitive phenotype or a strong export defect in most genetic backgrounds. The more common result is that deletion of secG causes only a mild export defect and does not result in conditional lethality. We propose that the role of SecG is not fundamental to the export process, but is merely auxiliary--as suggested previously by biochemical data--and is physiologically important only when cells are otherwise compromised.

Alleles↗

PubMed1998

Genetic analysis of an essential cytoplasmic domain of Escherichia coli SecY based on resistance to Syd, a SecY-interacting protein.

We previously described a dominant negative secY-d1 allele in Escherichia coli, whose product interferes with protein export, presumably by sequestering SecE, the stabilizing partner of SecY. Syd is the product of a multicopy suppressor of the secY-d1 phenotype, and its overproduction preferentially stabilizes the wild-type SecY protein. In contrast, overproduction of Syd is toxic to the secY24 mutant, which shows a partial defect in SecY-SecE interaction. We isolated Syd-resistant revertants from the secY24 mutant. Pseudo-reversions mapped to sites at or near the secY24 mutation site (Gly240-->Asp). The secY249 mutation (Ala249-->Val) intragenically suppressed Syd sensitivity, but not the temperature-sensitive Sec phenotype of the secY24 mutation. The SecY249 mutant protein shows a reduced capacity to be stabilized by Syd, suggesting that the mutation weakens the SecY-Syd interaction. The other two mutations changed residue 240 (the site of the secY24 alteration) to Asn (secY245) or Ala (secY241) and restored the ability of the cell to export protein. Although the secY245 mutant retained some sensitivity to Syd overproduction, the secY241 mutant was completely Syd-resistant. Furthermore, the secY241 mutation seemed to represent a "hyper reversion" with respect to the SecY-SecE interaction. Protein export in this mutant was no longer sensitive to SecY-d1. When the secY-d1 mutation was combined intragenically with secY241, the resulting double mutant gene (secY-d1-241) showed an increased ability to interfere with protein export. On the basis of our model for SecY-d1, these results suggest that the secY241 alteration enhances SecY-SecE interaction. These results indicate that residue 240 of SecY is crucial for the interaction between the cytosolic domains of SecY and SecE required for the establishment of the translocase complex.

ATP-Binding Cassette Transporters↗

PubMed2002

Isolation of fusion proteins containing SecY and SecE, components of the protein translocation complex from the halophilic archaeon Haloferax volcanii.

By exploiting the salt-insensitive interaction of the cellulose-binding domain (CBD) of the Clostridium thermocellum cellulosome with cellulose, purification of CBD-fused versions of SecY and SecE, components of the translocation apparatus of the halophilic archaeon Haloferax volcanii, was undertaken. Following transformation of Haloferax volcanii cells with CBD-SecY- or -SecE-encoding plasmids, cellulose-based purification led to the capture of stably expressed, membrane-bound 68 and 25 kDa proteins, respectively. Both fusion proteins were recognized by antibodies raised against the CBD. Thus, CBD-cellulose interactions can be employed as a salt-insensitive affinity purification system for the capture of complexes containing the Haloferax volcanii translocation apparatus components SecY and SecE.

Archaeal Proteins↗

PubMed2005

Gene replacement of adenylate kinase in the gram-positive thermophile Geobacillus stearothermophilus disrupts adenine nucleotide homeostasis and reduces cell viability.

Thermophilic bacteria are of great value for industry and research communities. Unfortunately, the cellular processes and mechanisms of these organisms remain largely understudied. In the present study, we investigate how the inactivation of adenylate kinase (AK) affects the adenine nucleotide homeostasis of a gram-positive moderate thermophile, Geobacillus stearothermophilus strain NUB3621-R. AK plays a major role in the adenine nucleotide homeostasis of living cells and has been shown to be essential for the gram-negative mesophile Escherichia coli. To study the role of AK in the maintenance of adenylate energy charge (EC) and cell viability of G. stearothermophilus, we generated a recombinant strain of this organism in which its endogenous gene coding for the essential protein adenylate kinase (AK) has been replaced with the adk gene from the mesophile Bacillus subtilis. PCR, DNA sequencing and Southern analysis were performed to confirm proper gene replacement and preservation of neighboring genes. The highest growing temperature for recombinant cells was almost 20 degrees C lower than for wild-type cells (56 vs. 75 degrees C). This temperature-sensitive phenotype was secondary to heat inactivation of B. subtilis AK, as evidenced by enzyme activity assays and EC measurements. At higher temperatures (65 degrees C), recombinant cells also had lower EC values (0.09) compared to wild-type cells (0.45), which reflects a disruption of adenine nucleotide homeostasis following AK inactivation.

Adenine Nucleotides↗

PubMed2005

Functional and structural characterization of the minimal Sec translocase of the hyperthermophile Thermotoga maritima.

The genome of the hyperthermophilic bacterium Thermotoga maritima contains the genes that encode core subunits of the protein translocase, a complex consisting of the molecular motor SecA and the protein conducting pore SecYE. In addition, we identified an erroneous sequence in the genome encoding for a putative secG gene. The genes of the T. maritima translocase subunits were overexpressed in Escherichia coli and purified to homogeneity. T. maritima SecA showed a basal thermostable ATPase activity that was stimulated up to 4-fold by phospholipids with an optimum at 74 degrees C. Membrane vesicles and proteoliposomes containing SecYE or SecYEG supported 2- to 4-fold stimulation of the precursor dependent SecA ATPase activity. Imaging of small two-dimensional crystals of the SecYE complex using electron microscopy showed square-shaped particles with a side-length of about 6 nm. These results demonstrate that in T. maritima a highly thermostable translocase complex is operational.

Adenosine Triphosphatases↗

PubMed1991

SecY is an indispensable component of the protein secretory machinery of Escherichia coli.

Using a reconstitution system for protein translocation, the involvement of SecY in the translocation of secretory proteins across the cytoplasmic membrane of Escherichia coli was studied. Anti-SecY antibodies raised against the N- and C-terminal sequences prevented the functional reconstitution of the translocation system. Depletion of SecY from the solubilized membrane preparation was performed by treatment with anti-SecY IgG, followed by removal of IgG with protein A-agarose. The SecY-depleted preparation was inactive as to functional reconstitution. However, reconstitution with it was demonstrated in the presence of a protein fraction, which was released from the anti-SecY immunoprecipitate upon addition of the SecY fragment used to raise the antibody. Reconstitution with the SecY-depleted membrane fraction was also demonstrated in the presence of a purified SecY preparation. OmpT proteinase specifically cleaved SecY in the solubilized membrane preparation. The cleavage was accompanied by a decrease in the reconstituted activity. Based on these findings we conclude that SecY is an indispensable component of the secretory machinery.

Amino Acid Sequence↗

PubMed1993

Overexpression of the ER-membrane protein P-450 CYP52A3 mimics sec mutant characteristics in Saccharomyces cerevisiae.

High expression of microsomal cytochrome P-450 CYP52A3 from Candida maltosa induces the formation of membrane stacks in Saccharomyces cerevisiae. Membrane proliferation is accompanied by coinduction of the ER proteins KAR2p and SEC61p and accumulation of precursor forms of proteins that have to translocate across the ER membrane (KAR2p, alpha factor). Cytosolic proteins (SSA1p and 2p) and mitochondrial proteins (CYT c1p and F1 beta p) are not affected. N-terminal truncated P-450 proteins remain in the cytoplasm and fail to induce membrane proliferation, KAR2p/SEC61p expression, and precursor accumulation. Membrane and precursor protein accumulation are typical features of sec mutants. We assume that the high amounts of P-450p block one or more factor(s) of the transport machinery and thereby cause the observed phenomena.

Amino Acid Sequence↗

PubMed1990

Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichia coli.

SecA is an essential component of the protein secretory machinery of Escherichia coli. SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.

Adenosine Triphosphatases↗

PubMed1990

SecY protein, a membrane-embedded secretion factor of E. coli, is cleaved by the ompT protease in vitro.

SecY is an integral membrane protein, spanning the cytoplasmic membrane of E. coli probably 10 times and required for efficient translocation of other proteins across the membrane. We report here that this protein can be specifically cleaved at the central region of the polypeptide after cell disruption, and cytoplasmic membrane preparations often contain a degradation product of SecY. This cleavage was ascribed to the action of the outer membrane-associated protease specified by the ompT gene, since the cleavage was not observed in ompT-defective mutants. Thus, we propose that an ompT mutant should be used for in vitro analysis of protein translocation and the SecY protein. We mutated the ompT gene by insertion of a kanamycin resistance determinant to facilitate strain construction by P1 transduction.

Bacterial Proteins↗

PubMed1991

SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer.

Size exclusion chromatography of the cytosolic fraction of SecA-overproducing cells of Escherichia coli suggested that SecA, an essential component of the secretory machinery, exists as an oligomer. The subunit structure of SecA was then studied using a purified specimen. Estimation of the molecular mass by means of ultracentrifugation and chemical crosslinking analysis revealed that SecA exists as a homodimer. The purified SecA was denatured in 6 M guanidine-HCl and renatured to a dimer, which was fully active in terms of translocation, even in the presence of 1 mM dithiothreitol. It is suggested that the dimeric structure is not critically maintained by disulfide bonding between the two subunits, each of which contains four cysteine residues.

Adenosine Triphosphatases↗