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Results for “SEC Translocation Channels”

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PubMed2001

Effects of lipids on the interaction of SecA with model membranes.

The effects of nonlamellar-prone lipids, diacylglycerol and phosphatidylethanolamine (PE), on the kinetic association of SecA with model membranes were examined by measuring changes in the intrinsic emission fluorescence with a stopped-flow apparatus. Upon interaction with standard liposomes composed of 50 mol% dioleolyphosphatidylcholine (DOPC) and 50 mol% of dioleoylphosphatidylglycerol (DOPG), the intrinsic fluorescence intensity of SecA was decreased after a lapse of time with a rate constant of 0.0049 s(-1). When the DOPC of the standard vesicles was gradually replaced with either dioeloyl PE (DOPE) or Escherichia coli (E. coli) PE, the rate constant increased appreciably as a function of PE concentration, in the order DOPE > E. coli PE. In addition, when the PE of E. coli PE/DOPG (50/50) vesicles was replaced with more than 5 mol% dioleoylglycerol (DOG), the rate constant further increased by 40%. The incorporation of nonlamellar-prone lipids in the vesicles also enhanced the binding of SecA to model membranes in the order DOPE > or = E. coli PE/DOG > E. coli PE > DOPC. These results provide the first kinetic evidence for the importance of nonlamellar-prone phospholipids for the association rate of SecA with membranes.

Adenosine Triphosphatases↗

PubMed1993

Sec-Y protein is localized in both the cytoplasmic and thylakoid membranes in the cyanobacterium Synechococcus PCC7942.

Members of the SecY protein family mediate protein export in bacterial cells. Southern analyses showed that secY is likely a single copy gene in the cyanobacterium Synechococcus PCC7942. Then the subcellular location of the cyanobacterial SecY protein was determined; i) antiserum raised against a fusion protein between the SecY fragment and maltose binding protein were used for immunoblotting of the membrane fractions, and ii) a modified SecY protein carrying the c-Myc peptide tag was expressed in the cyanobacterial cells, and the subcellular distribution of the SecY-c-Myc fusion protein was analyzed with the anti-c-Myc antibodies. The obtained results suggest that the SecY protein is localized in the thylakoid membrane as well as the cytoplasmic membrane; the SecY protein probably mediates protein translocation across both the cytoplasmic and thylakoid membranes in Synechococcus PCC7942.

Amino Acid Sequence↗

PubMed1993

Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein.

Treatment of SecA, an essential component of the protein translocation machinery of Escherichia coli, with a histidine-specific reagent, diethylpyrocarbonate, caused significant abolition of its translocation-coupled ATPase (translocation ATPase) activity, which requires a presecretory protein and membrane vesicles, whereas its endogenous ATPase (SecA-ATPase) activity was enhanced by a factor of 2. Diethylpyrocarbonate-treated SecA exhibited an absorption maximum at 240 nm due to the formation of N-carbethoxyhistidine. Upon the modification of about 5 of the total 22 histidine residues in the SecA molecule, both the abolition of its translocation ATPase activity and the enhancement of its SecA-ATPase activity occurred. Intact and modified SecA exhibited similar affinities for ATP, proOmpA and membranes, whereas Vmax of the translocation ATPase activity was significantly lower in the case of the modified SecA. ATP had no effect on the modification of SecA. Taken together, these results indicate that histidine residues susceptible to diethylpyrocarbonate are essential for the translocation ATPase, but not directly involved in the binding of ATP, proOmpA and membranes. A possible reason for the abolition of translocation ATPase is discussed.

Adenosine Triphosphatases↗

PubMed1993

Components of the protein translocation machinery in the thermophilic cyanobacterium Phormidium laminosum.

Cyanobacteria possess cytoplasmic membranes, which are common to all eubacteria, and an additional internal network of thylakoid membranes. We have investigated the distribution of the protein translocation machinery between the two membrane systems. We have demonstrated that leader peptidase activity exists in both membrane systems, suggesting that newly synthesized protein precursors are targeted directly rather than sorted after translocation. Southern blot analysis suggests that the leader peptidase is likely to be a single enzyme common to both membrane systems. We have also shown that cyanobacteria possess a single SecA homologue. These results indicate that the protein translocation machinery in the two membrane systems is likely to be the same and leaves open the question of what mechanism enables the cell to sort proteins between them.

Adenosine Triphosphatases↗

PubMed1994

Identification and characterization of the sec-A protein homologue in the cyanobacterium Synechococcus PCC7942.

The secA gene product mediates protein translocation across the cytoplasmic membrane in Escherichia coli. We have cloned a gene homologous to secA from the genome of the cyanobacterium Synechococcus PCC7942. The deduced amino acid sequence, 948 amino acids long, shows 43% homology with that of the E. coli secA and 47-48% homology with those of the algal plastid secA genes. Upon subcellular fractionation, the cyanobacterial SecA protein was mainly found as soluble homodimer in the cytosol, but the remaining small but distinct fraction was associated with both the cytoplasmic and thylakoid membranes. The SecA protein likely participates in protein translocation across both the cytoplasmic and thylakoid membranes in cyanobacterial cells.

Adenosine Triphosphatases↗

PubMed1994

Carboxy-terminal region of Escherichia coli SecA ATPase is important to promote its protein translocation activity in vivo.

The role of the carboxy-terminal region of E. coli SecA ATPase was investigated by using genetic methods to construct a truncated SecA protein missing the last 66 amino acid residues and by systematically substituting serine for each of four cysteine residues present in this protein. Truncation of SecA or alteration of any of the carboxy-terminal cysteine residues resulted in poor growth and a protein secretion defect, indicating that this region of SecA is important in its protein translocation activity. Biochemical analysis of the altered proteins revealed a modest increase in translocation ATPase activity, suggesting that the carboxy-terminal region of SecA may facilitate the coupling of its ATPase activity to cycles of protein translocation.

Adenosine Triphosphatases↗

PubMed1994

SecA of Escherichia coli traverses lipid bilayer of phospholipid vesicles.

SecA protein of Escherichia coli, when added externally to the vesicles composed of phosphatidylethanolamine, dioleoylphosphatidylglycerol and cardiolipin, was found to be fragmented by trypsin encapsulated within the vesicles. In the presence of ATP or its non-hydrolyzing analogue, ATP-gamma S, the number of fragments and extent of hydrolysis occurred much less than in the absence of these compounds. When ADP was added, however, the hydrolysis products were similar to those when no nucleotide was present. Quenching of SecA fluorescence by vesicle-entrapped iodide corroborated the digestion results. These experiments demonstrated that the SecA protein traverses the lipid bilayer and its membrane topology depends on the kind of nucleotide present.

Adenosine Triphosphatases↗

PubMed1995

Intracellular stability of alpha fragments of beta-galactosidase: effects of amino-terminally fused polypeptides.

Intracellular stability of alpha fragments of beta-galactosidase in Escherichia coli has been studied by pulse-chase/immunoprecipitation experiments. An alpha fragment encoded by the pUC118 vector was relatively stable with an estimated half-life of about 12 min at 37 degrees C, whereas another vector, pSTV28, encoded a less stable alpha fragment that had a different carboxy-terminal sequence. Stability of the fragment was found to be affected markedly by amino-terminal attachment of other sequences. An amino-terminal fusion of a sequence derived from cytoplasmic domain 4 of the SecY protein shortened the half-life of the alpha fragment to less than 1 min. In contrast, an amino-terminal sequence from the NusG protein had no apparent effect on the stability of the fragment. In a fusion protein in which the intact SecY protein was fused to the alpha fragment, stabilization of the SecY part by overproduction of the partner SecE protein resulted in an increased alpha complementation activity of beta-galactosidase. These results indicate that stability of alpha fragment can be dictated by the stability of the fused protein. The alpha fragment of beta-galactosidase, which is unique in that it is largely unstructured but can be "active" in alpha complementation, may be used as an in vivo indicator of stability of proteins attached to it.

Amino Acid Sequence↗

PubMed1995

Preferential interaction of Sec-G with Sec-E stabilizes an unstable Sec-E derivative in the Escherichia coli cytoplasmic membrane.

SecY, SecE and SecG form a membrane part of the protein translocation machinery. A SecG-overproducing plasmid was constructed by placing the secG gene under the control of the tac promoter. From the extent of SecG overproduction, the number of SecG molecules in one normal cell was estimated to be about 1,000, which is similar to those of SecY and SecE. Overproduction of SecG stabilized the overproduction of SecE-C, an unstable truncated derivative of SecE, as effectively as SecY does. SecG overproduction also stabilized the overproduction of SecY. However, the SecG-dependent stabilization of SecY was less potent than the SecE-dependent stabilization. These results indicate that SecG preferentially interacts with SecE, which associates with SecY, the SecG-SecE-SecY complex thus being formed in the cytoplasmic membrane.

Bacterial Proteins↗

PubMed1996

Cytochrome f encoded by the chloroplast genome is imported into thylakoids via the SecA-dependent pathway.

In vitro import of the precursor of tobacco cytochrome f, which is is encoded by the chloroplast genome, into isolated pea thylakoids was analyzed. Upon incubation with thylakoids and a stromal fraction, the precursor of cytochrome f was efficiently imported into thylakoids. The imported cytochrome f was tightly integrated into the thylakoid membrane. Insertion of cytochrome f into the thylakoid membrane was blocked by nigericin, sodium azide, and antibodies against pea chloroplast SecA. These results suggest that cytochrome f utilizes the bacterial-type SecA-dependent pathway to be imported into thylakoids.

Adenosine Triphosphatases↗

PubMed1996

The carboxyl-terminal region is essential for Sec-A dimerization.

SecA, comprising 901 amino acid residues, exists as a dimer. By means of size exclusion chromatography and chemical cross-linking analysis, five truncated SecA derivatives were examined to identify the region of SecA essential for dimer formation. Among them, only N95 (delta 832-901) retained SecA activity. N95 existed as a dimer, indicating that the carboxyl-terminal three cysteine residues are dispensable for physiological dimerization. Both N76 (delta 675-901) and N66 (delta 583-901) existed as monomers. Monomeric N76 was able to bind to ATP, indicating that the dimerization of SecA is not a prerequisite for ATP binding. However, the rate of ATP hydrolysis by N76 was 25% of that by SecA. C53 (delta 1-437) and C28 (delta 1-661) formed dimers irrespective of the presence or absence of 2-mercaptoethanol. C28, but not C53, also existed as an oligomer in the absence of 2-mercaptoethanol, suggesting that the 438-661 region present in C53 prevents intermolecular disulfide bond formation at the carboxyl-terminal cysteine residue. From these results, the region essential for the physiological dimer formation was concluded to be located in the 662-831 region of SecA.

Adenosine Triphosphatases↗

PubMed1997

RNA helicase activity of Escherichia coli SecA protein.

SecA protein of Escherichia coli (E. coli), an ATPase essential for the translocation of precursor proteins, was found to have an additional activity of RNA helicase. This RNA unwinding activity of SecA was tested with two kinds of RNA duplex with different predicted stability. Each of these duplexes is consisted of two strands of unequal length with single-stranded ends. The RNA helicase activity of SecA required ATP and divalent cations. Confirmation of this activity came from the inhibition of unwinding of the RNA duplex when SecA was preincubated with its own polyclonal antibody. The biological significance of the RNA helicase activity of E. coli SecA protein is discussed.

Adenosine Triphosphatases↗

PubMed1998

Molecular cloning and functional characterization of SecE of a marine bacterium, Vibrio alginolyticus.

SecE is an essential membrane component of the Escherichia coli protein translocation machinery, which utilizes ATP and the proton motive force as energy sources. The secE homologue of marine Vibrio alginolyticus, in which protein translocation requires ATP and the sodium motive force, was cloned, sequenced, and characterized. Unlike most SecE homologues found in various organisms, SecE of V. alginolyticus (Va-SecE) possesses three transmembrane sequences like E. coli SecE (Ec-SecE) and complements the DeltasecE mutation of E. coli. Alignment of the Ec-SecE and Va-SecE sequences revealed that the non-essential N-terminal half was less homologous than the essential C-terminal half between the two SecEs. The E. coli strain was able to grow and translocate the secretory protein in the complete absence of Ec-SecE when Va-SecE was expressed. The stabilization of SecY overproduction by Va-SecE was as effective as that by Ec-SecE, indicating that Va-SecE interacts with E. coli SecY despite the difference in energy requirement.

Amino Acid Sequence↗

PubMed1994

An ascidian homolog of SEC61 is expressed predominantly in epidermal cells of the embryo.

In eukaryotic cells, SEC61 protein is essential for protein translocation across the endoplasmic reticulum membrane. Because of the ubiquitous nature of the protein, the expression of the corresponding gene(s) during embryogenesis has not received much attention. We found that an ascidian homolog of SEC61 is expressed predominantly in embryonic epidermal cells. A cDNA clone for Halocynthia roretzi SEC61 (HRSEC61) gene contains a single open reading frame that encodes a polypeptide of 475 amino acids. The degree of amino-acid identity was 87% between the ascidian and the dog and 55% between the ascidian and yeast. The HRSEC61 gene was transcribed maternally and zygotically. A low level of the corresponding mRNAs, about 2.3 kb in length, was found in eggs and early embryos. Zygotic mRNAs appeared after the gastrula stage, and the accumulation of the mRNAs was maximal at the neurula and tailbud stages. In situ hybridization of whole-mount specimens demonstrated that, although maternal mRNAs were distributed evenly in eggs and early embryos, the occurrence of zygotic mRNAs was predominant in differentiating epidermal cells. The predominant expression of HRSEC61 in embryonic epidermal cells was determined using cleavage-arrested embryos; those at the 16- or 32-cell stage expressed HRSEC61 in the presumptive epidermal cells of the animal hemisphere.

Amino Acid Sequence↗

PubMed1996

The preprotein translocase of the inner mitochondrial membrane: evolutionary conservation of targeting and assembly of Tim17.

The preprotein translocase of the inner mitochondrial membrane has only been described in Saccharomyces cerevisiae to date. We report that the essential subunit Tim17 is highly conserved in evolution. The targeting and assembly of yeast Tim17 as well as that of human and Drosophila melanogaster Tim17 were characterized with isolated yeast mitochondria. Targeting signals in the mature protein direct the Tim17 precursors to the receptor Tom70 on the mitochondrial surface. In a membrane potential-dependent step the precursors insert into the inner membrane, adopt a characteristic topology and assemble with Tim23. The mechanisms of targeting and assembly were indistinguishable between the Tim17s from distinct organisms, indicating a high evolutionary conservation.

Adenosine Triphosphatases↗

PubMed1997

Dual regulation of Escherichia coli secA translation by distinct upstream elements.

The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was investigated using a combination of genetic and biochemical approaches. Oligonucleotide-directed deletion and point mutagenesis was used to show that only the last quarter of the upstream gene, geneX, and the geneX-secA intergenic are essential for proper regulation. This region previously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude the secA Shine-Dalgarno sequence. Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-repression by SecA protein, but retained a normal pattern of derepression of secA expression during a protein export block. The introduction of compensatory mutations into helix II that were predicted to restore base-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation in vivo. In contrast, mutations in the lower portion of helix I decreased secA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export block. In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore proper secA regulation, indicating that specific nucleotides in this region are required for normal secA regulation. Primer-extension inhibition (toeprint) analysis with 30 S ribosoma subunits, tRNAMet, and a model segment of geneX-secA RNA carrying the relevant mutations was used to show that mutations that destabilized helix II increased ribosome binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site. Our results suggest strongly that there is a system of dual regulation of secA translation, whereby helix I serves as an activator element while helix II serves as a repressor element.

Adenosine Triphosphatases↗

PubMed1997

Identification of elements on GeneX-secA RNA of Escherichia coli required for SecA binding and secA auto-regulation.

The protein translocation ATPase of Escherichia coli, SecA protein, auto-regulates its translation by binding to its translation initiation region in geneX-secA mRNA. To analyze this regulation further the secondary structure of this portion of geneX-secA RNA was investigated utilizing structure-specific nucleases and chemical probing approaches. The results of this analysis were consistent with the existence of two adjacent helices, helix I and the lower portion of helix II, whose function in secA activation and repression, respectively, has been demonstrated. Binding of SecA protein to geneX-secA RNA or various mutant derivatives of this RNA was studied by measurement of affinity constants, RNA footprint analysis, and quantitation of auto-repression in vivo. This analysis showed that the SecA-binding site in geneX-secA RNA was remarkably large spanning a region of 96 nucleotides including a 3' portion of helix II, the secA translation initiation region and distal sequences. From the size of the SecA-binding site and the plasticity of its response to mutational alteration, it is suggested that SecA protein contains two distinct RNA-binding sites. Finally, it was shown that SecA binding was not sufficient to promote auto-regulation and that sequences both upstream (helix I) and within the binding site can contribute to auto-regulation without affecting SecA-binding affinity.

Adenosine Triphosphatases↗

PubMed1998

Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: an implication from the interference by a mutant form of a new substrate protein, YccA.

Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here, we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein. From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.

ATP-Dependent Proteases↗