Biochemical characterization of the SecA protein of <i>Streptomyces lividans</i>

Blanco, Jorge; Driessen, Arnold J. M.; Coque, Juan José R.; Martı́n, Juan F.

doi:10.1046/j.1432-1327.1998.2570472.x

Cited by 12 publications

(13 citation statements)

References 52 publications

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“…This indicates that both nucleotides bind cpSecA and that ATP can displace mant-ADP in a dose-dependent manner indicative of a specific interaction with nucleotide binding sites. Consistent with this observation, cpSecA was found to have substantial ATPase activity in aqueous solution (90 pmol min Ϫ1 g SecA Ϫ1 ), which is further enhanced in the presence of lipid vesicles (120 pmol min Ϫ1 g SecA Ϫ1 ), as has been observed for SecA from other species (2,17).…”

supporting

confidence: 80%

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

et al. 2007

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Like prokaryotic Sec-dependent protein transport, chloroplasts utilize SecA. However, we observe distinctive requirements for the stimulation of chloroplast SecA ATPase activity; it is optimally stimulated in the presence of galactolipid and only a small fraction of anionic lipid and by Sec-dependent thylakoid signal peptides but not Escherichia coli signal peptides.The chloroplast Sec pathway for protein transport is particularly interesting because it sits at the evolutionary crossroads between eukaryotic systems which have Sec-dependent pathways that do not involve SecA (e.g., endoplasmic reticulum [ER] transport) and prokaryotic Sec systems that do require SecA. In bacteria, the components of this pathway include the SecYEG complex, which constitutes the translocation channel (8, 11), and SecA, an ATPase that powers the translocation of the polypeptide across the cytoplasmic membrane (16,31,32). In Escherichia coli, the efficiency of protein translocation is directly proportional to the amount of anionic phospholipids (17,20,25) and this reflects the effect of these lipids on SecA ATPase activity (19,29). In plants, genes encoding chloroplast homologues of SecY (15) and SecE (10) have been identified, as has a SecA homologue from several chloroplast sources (22,24,26,28). The lipid composition of the thylakoid membrane is dominated by the neutral galactolipid digalactosyldiacylglycerol (DGDG) (70 to 80%) and anionic dioleylphosphatidylglycerol (DOPG) (30). The extent to which these lipids influence the activity of the thylakoid transport machinery is not known.Sec-dependent proteins that cross the eukaryotic ER or the bacterial cytoplasmic membrane are synthesized with a single amino-terminal signal peptide whose highly hydrophobic nature plays a critical role in facilitating protein transport (5) through interactions with SecA (4). Proteins destined for the Sec pathway in the chloroplast thylakoid differ in that they are synthesized with presequences that are bipartite; the most aminoterminal portion corresponds to a transit sequence for passage through the chloroplast envelope and the second region is reminiscent of bacterial and ER signals. Does chloroplast SecA (cpSecA) directly interact with this signal, and is it tailored to discriminate thylakoid Sec signals from the variety of targeting signals for other chloroplast transport pathways?Here, we address these issues by using cpSecA ATPase activity as an earmark of ligand interactions. In marked contrast to E. coli SecA, cpSecA ATPase activity is enhanced by a high concentration of DGDG and only a small amount of DOPG. Furthermore, cpSecA ATPase activity is preferentially stimulated by weakly hydrophobic chloroplast, but not bacterial, Sec signal peptides.Helical content and ATPase activity of cpSecA is retained in lipid vesicles. To set the stage for examining the preferences of cpSecA for two key ligands, lipids and signal peptides, we first established that SecA retained secondary structural elements and ATPase activity upon lipid vesicle integration...

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supporting

confidence: 80%

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

et al. 2007

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“…Further evidence that functional signal peptides specifically bind SecA in both aqueous solution and a lipid environment is provided by an assay based on the sensitivity of SecA to V8 protease (9,28). In general, digestion of the 102-kDa SecA by V8 involves the initial loss of a carboxyl-terminal fragment, leaving a 95-kDa truncated form of SecA (9), which is subsequently completely digested to low molecular weight fragments after 2-3 h of proteolysis (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…V8 Proteolysis of SecA-Proteolytic digestions of SecA with V8 protease were conducted at 37°C, essentially as described (9,28). All reaction mixtures (total volume, 25 l) contained SecA (10 g), 50 mM Tris-HCl (pH 8), 50 mM KCl, 5 mM MgCl 2 , and 1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

Signal Peptide Determinants of SecA Binding and Stimulation of ATPase Activity

Wang

Miller

Kendall

2000

Journal of Biological Chemistry

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A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.

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“…We propose that FbpB-ЈPhoA and Rv1566c-ЈPhoA are two such proteins, since SecA2 participates, but dos not exclusively act, in their export. In E. coli, B. subtilis, and Streptomyces lividans, SecA has been shown to exist as a dimer (1,3,13,47). Therefore, it is also possible that SecA1 and SecA2 form mixed dimers and that this species is important in the recognition of certain substrates.…”

Section: Discussionmentioning

confidence: 99%

Two Nonredundant SecA Homologues Function in Mycobacteria

et al. 2001

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The proper extracytoplasmic localization of proteins is an important aspect of mycobacterial physiology and the pathogenesis of Mycobacterium tuberculosis. The protein export systems of mycobacteria have remained unexplored. The Sec-dependent protein export pathway has been well characterized in Escherichia coli and is responsible for transport across the cytoplasmic membrane of proteins containing signal sequences at their amino termini. SecA is a central component of this pathway, and it is highly conserved throughout bacteria. Here we report on an unusual property of mycobacterial protein export-the presence of two homologues of SecA (SecA1 and SecA2). Using an allelic-exchange strategy in Mycobacterium smegmatis, we demonstrate that secA1 is an essential gene. In contrast, secA2 can be deleted and is the first example of a nonessential secA homologue. The essential nature of secA1, which is consistent with the conserved Sec pathway, leads us to believe that secA1 represents the equivalent of E. coli secA. The results of a phenotypic analysis of a ⌬secA2 mutant of M. smegmatis are presented here and also indicate a role for SecA2 in protein export. Based on our study, it appears that SecA2 can assist SecA1 in the export of some proteins via the Sec pathway. However, SecA2 is not the functional equivalent of SecA1. This finding, in combination with the fact that SecA2 is highly conserved throughout mycobacteria, suggests a second role for SecA2. The possibility exists that another role for SecA2 is to export a specific subset of proteins.

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Biochemical characterization of the SecA protein of Streptomyces lividans

Cited by 12 publications

References 52 publications

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

Chloroplast SecA and Escherichia coli SecA Have Distinct Lipid and Signal Peptide Preferences

Signal Peptide Determinants of SecA Binding and Stimulation of ATPase Activity

Two Nonredundant SecA Homologues Function in Mycobacteria

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