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PubMed2002

The ATPase domain of SecA can form a tetramer in solution.

Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=[monomer](4)/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.

Adenosine Triphosphatases↗

PubMed1994

The SecY protein family: comparative analysis and phylogenetic relationships.

We have cloned and sequenced the plastome encoded secY homologue of the cryptomonad alga Pyrenomonas salina. In this study we have carried out a comparative analysis of all but one fully sequenced proteins of the SecY family. We present an alignment of 16 SecY family proteins, containing family signatures, putative transmembrane domains, consensus sequence, and conservation grade. A phylogenetic tree derived from the conserved blocks of the alignment reveals the relationships among this protein family. The tree shows division into two broad subfamilies, one consisting of prokaryotic and plastidal sequences and the other of eukaryotic as well as archaeal sequences.

Amino Acid Sequence↗

PubMed1998

Expression, purification, and characterization of Sss1p, an essential component of the yeast Sec61p protein translocation complex.

Sss1p, a 8.9-kDa membrane protein, is an essential component of the protein translocation complex involved in the transport of secretory proteins across the Saccharomyces cerevisiae endoplasmic reticulum membrane. In order to determine the high resolution structure of Sss1p by NMR, we have undertaken its overexpression and purification. We first inserted the yeast SSS1 gene into the pGEX-2T plasmid expression vector. Sss1p was expressed as fusions with Schistosoma japonica glutathione S-transferase (GST-Sss1p) in MC1061 Escherichia coli cells. Maximum yield of GST-Sss1p was obtained from cells harvested 2 h after induction at 37 degreesC in Luria broth medium. GST-Sss1p was found associated predominantly with the membrane pool and was readily extracted with Triton X-100. Detergent-solubilized GST-Sss1p was isolated by adsorption on glutathione-agarose beads. Sss1p was released from its GST carrier by cleavage with thrombin and its recovery was maximized by addition of dodecyl maltoside. Desorbed Sss1p was loaded on a high-performance liquid chromatography hydroxyapatite column equilibrated in phosphate buffer supplemented with dodecyl maltoside and the fractions containing Sss1p were subsequently purified to homogeneity by reverse-phase chromatography on a C4 column. The entire purification protocol can be completed in 5-6 h and yields about 0.4 mg of Sss1p per gram of transformed cells. CD and preliminary 1H NMR experiments show that purified Sss1p solubilized in SDS micelles is very stable and adopts a helical secondary structure.

Amino Acid Sequence↗

PubMed2005

Recognition and delivery of ERAD substrates to the proteasome and alternative paths for cell survival.

Endoplasmic reticulum-associated protein degradation (ERAD) is a protein quality control mechanism that minimizes the detrimental effects of protein misfolding in the secretory pathway. Molecular chaperones and ER lumenal lectins are essential components of this process because they maintain the solubility of unfolded proteins and can target ERAD substrates to the cytoplasmic proteasome. Other factors are likely required to aid in the selection of ERAD substrates, and distinct proteinaceous machineries are required for substrate retrotranslocation/dislocation from the ER and proteasome targeting. When the capacity of the ERAD machinery is exceeded or compromised, multiple degradative routes can be enlisted to prevent the detrimental consequences of ERAD substrate accumulation, which include cell death and disease.

Animals↗

PubMed1995

Characterization of plastocyanin from the cyanobacterium Phormidium laminosum: copper-inducible expression and SecA-dependent targeting in Escherichia coli.

Plastocyanin from the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and the gene cloned and sequenced. The derived amino acid sequence indicates that the plastocyanin protein is initially synthesized with an N-terminal leader sequence of 34 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a hydrophobic region and a cleavage site, which are characteristic both of higher-plant chloroplast thylakoid transfer domains and of bacterial leader peptides. The petE gene and flanking regions have been cloned in Escherichia coli, and the plastocyanin protein is expressed and directed to the periplasmic space, with concomitant processing to the mature form. Targeting to the periplasm and processing of the plastocyanin protein in E. coli appears to be dependent on components of the Sec apparatus, since the unprocessed precursor accumulates in the cytoplasm of a secA mutant. Expression of plastocyanin in E. coli is copper-inducible and apparently controlled at the level of transcription, leading to the conclusion that copper-regulated promoters exist in the regions flanking the gene and are recognized in a heterologous system. Possible implications for gene expression and protein targeting in the cyanobacterium are discussed.

Adenosine Triphosphatases↗

PubMed1996

A model for the evolution of the plastid sec apparatus inferred from secY gene phylogeny.

Plastids possess a bacteria-like sec apparatus that is involved in protein import into the thylakoid lumen. We have analyzed one of the genes essential for this process, secY. A secY gene from the unicellular red alga Cyanidium caldarium was found to be transcriptionally active, demonstrating for the first time that secY is functional in a plastid. Unlike the situation seen in bacteria the C. caldarium gene is transcribed monocistronically, despite the fact that it is part of a large ribosomal gene cluster that resembles bacterial spc operons. A molecular phylogeny is presented for 8 plastid-encoded secY genes, four of which have not been published yet. In this analysis plastid secY genes fall into two classes. One of these, comprising of genes from multicellular red algae and Cryptophyta, clusters in a neighbour-joining tree with a cyanobacterial counterpart. Separated from the aforesaid are secY genes from Chromophyta, Glaucocystophyta and a unicellular red alga. All plastid and cyanobacterial sequences are located on the same branch, separated from bacterial homologues. We postulate that the two classes of secY genes are paralogous, i.e. their gene products are involved in different protein translocation processes. Based on this assumption a model for the evolution of the plastid sec apparatus is presented.

Cloning, Molecular↗

PubMed1991

Identification of a gene fragment which codes for the 364 amino-terminal amino acid residues of a SecA homologue from Bacillus subtilis: further evidence for the conservation of the protein export apparatus in gram-positive and gram-negative bacteria.

A DNA fragment that codes for the 364 amino-terminal amino acid residues of a putative Bacillus subtilis SecA homologue has been cloned using the Escherichia coli secA gene as a probe. The deduced amino acid sequence showed 58% identity to the amino-terminus of the E. coli SecA protein. A DNA fragment which codes for 275 amino-terminal amino acid residues of the B. subtilis SecA homologue was expressed in E. coli and the corresponding gene product was shown to be recognized by anti-E. coli SecA antibodies. This polypeptide, although only about 30% the size of the E. coli SecA protein, also restored growth of E. coli MM52 (secAts) at the non-permissive temperature and the translocation defect of proOmpA in this mutant was relieved to a substantial extent.

Adenosine Triphosphatases↗

PubMed1993

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus.

Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli secA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secA Y may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.

Adenosine Triphosphatases↗

PubMed1994

Cloning and characterization of a secY homolog from Chlamydia trachomatis.

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-alpha ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50,195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SecY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.

Amino Acid Sequence↗

PubMed1992

Suppression of the growth and export defects of an Escherichia coli secA(Ts) mutant by a gene cloned from Bacillus subtilis.

A gene library of Bacillus subtilis chromosomal DNA was screened for genes capable of reverting the growth defects of the Escherichia coli secA51(Ts) mutant at 42 degrees C. A B. subtilis gene, designated csaA, was found to phenotypically suppress not only the growth defects of the E. coli mutant, but also to relieve the detrimental accumulation of precursors of exported proteins. The csaA gene encoded a protein of 15 kDa (137 amino acids) and was likely to be the distalmost member of an operon. No similarity to csaA was found among DNA or protein sequences deposited in databases. In contrast to other homologous or heterologous suppressors of the E. coli secA51(Ts) mutation, the csaA gene did not exert pleiotropic effects on either the E. coli secY24(Ts) or lep9(Ts) mutations. However, it restored the ability of a SecB-deficient mutant to grow on complex medium. It is proposed that CsaA serves as a molecular chaperone for exported proteins or alternatively acts by stabilizing the SecA protein.

Adenosine Triphosphatases↗

PubMed1992

Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family.

SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli. We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus. The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments. Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes. Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains. It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.

Amino Acid Sequence↗

PubMed1994

Genetic analysis of SecY: additional export-defective mutations and factors affecting their phenotypes.

A number of secY mutants of Escherichia coli showing protein export defects were isolated by a combination of localized mutagenesis and secA-lacZ screening. Most of them were cold sensitive and contained single base substitutions in secY leading to amino acid replacements in various parts of the SecY protein, mainly in the cytoplasmic and the transmembrane domains. A temperature-sensitive mutant with an export defect had the same base substitution as secY24, which was characterized previously. Many cold-sensitive secY mutants exhibited rapid responses to temperature lowering but their apparent defects varied at the permissive temperature. Others exhibited delayed responses to the temperature shift. Some secY mutations, including secY39, interfered with protein export when expressed from a multicopy plasmid, even in the presence of wild-type secY on the chromosome. Such "dominant negative" mutations, including secY-d1, which was studied previously, were all located in either cytoplasmic domain 5 or 6, which is consistent with our previous proposal that the C-terminal region of SecY is important for its function as a protein translocator. We also studied the phenotypes of strains in which one of the secY mutations was combined with the components of the secD operon. Overexpression of secD partially suppressed the secY39 mutation, while overexpression of secF exacerbated the export defects of secY122 and secY125 mutations. Overexpression of "yajC", located within the secD operon, suppressed secY-d1. Although yajC itself proved to be dispensable, its disruption impaired the growth of the secY39 mutant at 42 degrees C. These observations suggest that SecY interacts with SecD, SecF, and the product of yajC.

Amino Acid Sequence↗

PubMedDate not supplied

cDNA cloning of a Sec61 homologue from the cryptomonad alga Pyrenomonas salina.

Sec61 is an endoplasmic reticulum transmembrane protein involved in the process of translocation of proteins across this membrane. To-date, the only cloned genes for Sec61 are derived from mammals and yeast. In this paper, we present the first full-length cDNA from a sec61 gene of a plant cell. Comparison of the predicted protein sequence with all known Sec61 proteins, as well as with the bacterial/plastome-encoded homologue SecY, demonstrates a high degree of similarity among the SecY/Sec61 family.

Amino Acid Sequence↗

PubMed1992

Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation.

SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.

Adenosine Triphosphatases↗

PubMed1990

Structure, function, and biogenesis of SecY, an integral membrane protein involved in protein export.

The E. coli secY (prlA) gene, located in the operator-distal part of the spc ribosomal protein operon, codes for an integral membrane protein, SecY. The phenotypes of temperature-sensitive and cold-sensitive mutations in secY suggest that the SecY protein plays an essential role in vivo to facilitate protein translocation, whereas the prlA mutations in this gene suggest that SecY may interact with the signal sequence of translocating polypeptides. SecY contains most probably six cytoplasmic and five periplasmic domains, as well as 10 transmembrane segments. Such membrane-embedded structure may confer the SecY protein a "translocator" function, in which it provides a protein-aceous pathway for passage of secreted as well as membrane proteins. Results obtained by in vitro analyses of the translocation reactions, as well as some new phenotypes of the secY mutants, are consistent with this notion. Possible interaction of SecY with other secretion and chaperone-like factors is also discussed.

Bacterial Proteins↗

PubMed1994

Rapid purification of native SecA from Escherichia coli: development of a new affinity chromatography procedure.

The SecA protein occupies a pivotal position in the public protein export pathway in Escherichia coli. The multifunctional SecA protein recognizes cytoplasmic factors associated with export including the presecretory protein and targets the complex to the inner membrane, where it acts in the early stages of protein translocation. The ability of SecA to bind ATP was the basis for the development of a novel, rapid purification scheme involving a single chromatographic step. Affinity chromatography was carried out on Red Sepharose CL-6B. The SecA present in crude extracts of E. coli binds strongly to this dye-ligand matrix, and active protein was purified to greater than 90% homogeneity. The protein isolated by this procedure retained the previously described ATPase and RNA-binding activities of SecA. This approach should permit the rapid purification of SecA homologs from a variety microorganisms.

Adenosine Triphosphatases↗

PubMed2001

Transport of proteins into mitochondria.

Most mitochondrial proteins are nuclear-encoded and synthesised as preproteins on polysomes in the cytosol. They must be targeted to and translocated into mitochondria. Newly synthesised preproteins interact with cytosolic factors until their recognition by receptors on the surface of mitochondria. Import into or across the outer membrane is mediated by a dynamic protein complex coined the translocase of the outer membrane (TOM). Preproteins that are imported into the matrix or inner membrane of mitochondria require the action of one of two translocation complexes of the inner membrane (TIMs). The import pathway of preproteins is predetermined by their intrinsic targeting and sorting signals. Energy input in the form of ATP and the electrical gradient across the inner membrane is required for protein translocation into mitochondria. Newly imported proteins may require molecular chaperones for their correct folding.

Adenosine Triphosphatases↗

PubMed2002

SecB, one small chaperone in the complex milieu of the cell.

SecB is only one of a plethora of cytosolic chaperones in E. coli whose common property is that they bind nonnative proteins. It plays a crucial role during protein export via the general secretory pathway by modulating the partitioning of precursors between folding or aggregation and delivery to the membrane-bound translocation apparatus. In this latter role SecB demonstrates specific binding to a unique partner, SecA. SecB has the potential to participate in functions outside of export acting as a general nonspecific chaperone to provide buffering capacity of the nonnative state of proteins in the cytosolic pool. We discuss the interactions of SecB with its many binding partners in light of its recently determined structure, emphasizing both kinetic and thermodynamic parameters.

Adenosine Triphosphatases↗