Abstract
Escherichia coli azi mutants, whose growth is resistant to millimolar concentrations of sodium azide, were among the earliest E. coli mutants isolated. Genetic complementation, mapping, and DNA sequence analysis now show that these mutations are alleles of the secA gene, which is essential for protein export across the E. coli plasma membrane. We have found that sodium azide is an extremely rapid and potent inhibitor of protein export in vivo and that azi mutants are more resistant to such inhibition. Furthermore, SecA-dependent in vitro protein translocation and ATPase activities are inhibited by sodium azide, and SecA protein prepared from an azi mutant strain is more resistant to such inhibition. These studies point to the utility of specific inhibitors of protein export, such as sodium azide, in facilitating the dissection of the function of individual components of the protein export machinery.
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bhatnagar S. K., Bessman M. J. Studies on the mutator gene, mutT of Escherichia coli. Molecular cloning of the gene, purification of the gene product, and identification of a novel nucleoside triphosphatase. J Biol Chem. 1988 Jun 25;263(18):8953–8957. [PubMed] [Google Scholar]
- Botstein D., Maurer R. Genetic approaches to the analysis of microbial development. Annu Rev Genet. 1982;16:61–83. doi: 10.1146/annurev.ge.16.120182.000425. [DOI] [PubMed] [Google Scholar]
- Cabelli R. J., Chen L., Tai P. C., Oliver D. B. SecA protein is required for secretory protein translocation into E. coli membrane vesicles. Cell. 1988 Nov 18;55(4):683–692. doi: 10.1016/0092-8674(88)90227-9. [DOI] [PubMed] [Google Scholar]
- Chen L., Tai P. C. ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384–4388. doi: 10.1073/pnas.82.13.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chin D. T., Goff S. A., Webster T., Smith T., Goldberg A. L. Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La. J Biol Chem. 1988 Aug 25;263(24):11718–11728. [PubMed] [Google Scholar]
- Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
- Crooke E., Guthrie B., Lecker S., Lill R., Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. doi: 10.1016/0092-8674(88)90115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cunningham K., Lill R., Crooke E., Rice M., Moore K., Wickner W., Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. doi: 10.1002/j.1460-2075.1989.tb03457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cunningham K., Wickner W. Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8630–8634. doi: 10.1073/pnas.86.22.8630. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deshaies R. J., Koch B. D., Werner-Washburne M., Craig E. A., Schekman R. A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor polypeptides. Nature. 1988 Apr 28;332(6167):800–805. doi: 10.1038/332800a0. [DOI] [PubMed] [Google Scholar]
- Fandl J. P., Tai P. C. Biochemical evidence for the secY24 defect in Escherichia coli protein translocation and its suppression by soluble cytoplasmic factors. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7448–7452. doi: 10.1073/pnas.84.21.7448. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gardel C., Benson S., Hunt J., Michaelis S., Beckwith J. secD, a new gene involved in protein export in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1286–1290. doi: 10.1128/jb.169.3.1286-1290.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
- Jones C. A., Holland I. B. Inactivation of essential division genes, ftsA, ftsZ, suppresses mutations at sfiB, a locus mediating division inhibition during the SOS response in E. coli. EMBO J. 1984 May;3(5):1181–1186. doi: 10.1002/j.1460-2075.1984.tb01948.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumamoto C. A., Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. doi: 10.1128/jb.154.1.253-260.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumamoto C. A., Chen L., Fandl J., Tai P. C. Purification of the Escherichia coli secB gene product and demonstration of its activity in an in vitro protein translocation system. J Biol Chem. 1989 Feb 5;264(4):2242–2249. [PubMed] [Google Scholar]
- LEDERBERG J. The selection of genetic recombinations with bacterial growth inhibitors. J Bacteriol. 1950 Feb;59(2):211–215. doi: 10.1128/jb.59.2.211-215.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lill R., Cunningham K., Brundage L. A., Ito K., Oliver D., Wickner W. SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli. EMBO J. 1989 Mar;8(3):961–966. doi: 10.1002/j.1460-2075.1989.tb03458.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lutkenhaus J. F., Wolf-Watz H., Donachie W. D. Organization of genes in the ftsA-envA region of the Escherichia coli genetic map and identification of a new fts locus (ftsZ). J Bacteriol. 1980 May;142(2):615–620. doi: 10.1128/jb.142.2.615-620.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Noumi T., Maeda M., Futai M. Mode of inhibition of sodium azide on H+-ATPase of Escherichia coli. FEBS Lett. 1987 Mar 23;213(2):381–384. doi: 10.1016/0014-5793(87)81526-0. [DOI] [PubMed] [Google Scholar]
- Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
- Oliver D. B., Cabelli R. J., Jarosik G. P. SecA protein: autoregulated initiator of secretory precursor protein translocation across the E. coli plasma membrane. J Bioenerg Biomembr. 1990 Jun;22(3):311–336. doi: 10.1007/BF00763170. [DOI] [PubMed] [Google Scholar]
- Riggs P. D., Derman A. I., Beckwith J. A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics. 1988 Apr;118(4):571–579. doi: 10.1093/genetics/118.4.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rollo E. E., Oliver D. B. Regulation of the Escherichia coli secA gene by protein secretion defects: analysis of secA, secB, secD, and secY mutants. J Bacteriol. 1988 Jul;170(7):3281–3282. doi: 10.1128/jb.170.7.3281-3282.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rothblatt J. A., Meyer D. I. Secretion in yeast: translocation and glycosylation of prepro-alpha-factor in vitro can occur via an ATP-dependent post-translational mechanism. EMBO J. 1986 May;5(5):1031–1036. doi: 10.1002/j.1460-2075.1986.tb04318.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schatz P. J., Riggs P. D., Jacq A., Fath M. J., Beckwith J. The secE gene encodes an integral membrane protein required for protein export in Escherichia coli. Genes Dev. 1989 Jul;3(7):1035–1044. doi: 10.1101/gad.3.7.1035. [DOI] [PubMed] [Google Scholar]
- Schmidt M. G., Oliver D. B. SecA protein autogenously represses its own translation during normal protein secretion in Escherichia coli. J Bacteriol. 1989 Feb;171(2):643–649. doi: 10.1128/jb.171.2.643-649.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmidt M. G., Rollo E. E., Grodberg J., Oliver D. B. Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli. J Bacteriol. 1988 Aug;170(8):3404–3414. doi: 10.1128/jb.170.8.3404-3414.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shiba K., Ito K., Yura T., Cerretti D. P. A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant. EMBO J. 1984 Mar;3(3):631–635. doi: 10.1002/j.1460-2075.1984.tb01859.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
- Tribhuvan R. C., Pilgaokar A. K., Pradhan D. S., Sreenivasan A. Effect of phenethyl alcohol on induction of alkaline phosphatase in Escherichia coli. Biochem Biophys Res Commun. 1970 Oct 9;41(1):244–250. doi: 10.1016/0006-291x(70)90495-x. [DOI] [PubMed] [Google Scholar]
- Watanabe M., Blobel G. Cytosolic factor purified from Escherichia coli is necessary and sufficient for the export of a preprotein and is a homotetramer of SecB. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2728–2732. doi: 10.1073/pnas.86.8.2728. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss J. B., Ray P. H., Bassford P. J., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. doi: 10.1073/pnas.85.23.8978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yura T., Wada C. Phenethyl alcohol resistance in Escherichia coli. I. Resistance of strain C600 and its relation to azide resistance. Genetics. 1968 Jun;59(2):177–190. doi: 10.1093/genetics/59.2.177. [DOI] [PMC free article] [PubMed] [Google Scholar]