Abstract
The secretion in Escherichia coli of a C-terminally truncated periplasmic enzyme from Salmonella typhimurium, the glpQ-encoded glycerolphosphate phosphodiesterase, was studied. Plasmid pRH100, carrying the truncated glpQ gene, directs the synthesis of a 30,000-molecular-weight (30 K) protein that is processed to a mature 27.5 K protein. (The mature wild-type protein is a 38 K protein.) The truncated protein is not released into the periplasm but remains membrane associated, although it becomes protease sensitive after conversion of cells to spheroplasts. The presence of pRH100 strongly reduces the amount of some other proteins in the periplasm, including the maltose- and ribose-binding proteins. The reduction does not occur at the level of transcription or early translation, as shown by lacZ fusions to the gene coding for the structural gene of the maltose-binding protein. Outer membrane proteins are not affected. A hydroxylamine-induced mutation in the sequence of glpQ corresponding to the mature polypeptide overcomes the inhibitory effect of pRH100. The mutated gene no longer directs the synthesis of the 30/27.5 K protein but directs that of a new 19 K protein which is not membrane bound. We propose that sorting signals in the mature GIpQ protein are necessary for effective translocation to the periplasm and that the C-terminal third of the protein is essential for release into the periplasm.
Full text
PDF






Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alton N. K., Vapnek D. Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature. 1979 Dec 20;282(5741):864–869. doi: 10.1038/282864a0. [DOI] [PubMed] [Google Scholar]
- Bassford P. J., Jr, Silhavy T. J., Beckwith J. R. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979 Jul;139(1):19–31. doi: 10.1128/jb.139.1.19-31.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Benson S. A., Bremer E., Silhavy T. J. Intragenic regions required for LamB export. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3830–3834. doi: 10.1073/pnas.81.12.3830. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chung C. H., Waxman L., Goldberg A. L. Studies of the protein encoded by the lon mutation, capR9, in Escherichia coli. A labile form of the ATP-dependent protease La that inhibits the wild type protease. J Biol Chem. 1983 Jan 10;258(1):215–221. [PubMed] [Google Scholar]
- Dougan G., Sherratt D. The transposon Tn1 as a probe for studying ColE1 structure and function. Mol Gen Genet. 1977 Mar 7;151(2):151–160. doi: 10.1007/BF00338689. [DOI] [PubMed] [Google Scholar]
- Emr S. D., Hall M. N., Silhavy T. J. A mechanism of protein localization: the signal hypothesis and bacteria. J Cell Biol. 1980 Sep;86(3):701–711. doi: 10.1083/jcb.86.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ferro-Novick S., Honma M., Beckwith J. The product of gene secC is involved in the synthesis of exported proteins in E. coli. Cell. 1984 Aug;38(1):211–217. doi: 10.1016/0092-8674(84)90542-7. [DOI] [PubMed] [Google Scholar]
- Hengge R., Larson T. J., Boos W. sn-Glycerol-3-phosphate transport in Salmonella typhimurium. J Bacteriol. 1983 Jul;155(1):186–195. doi: 10.1128/jb.155.1.186-195.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henning U., Cole S. T., Bremer E., Hindennach I., Schaller H. Gene fusions using the ompA gene coding for a major outer-membrane protein of Escherichia coli K12. Eur J Biochem. 1983 Nov 2;136(2):233–240. doi: 10.1111/j.1432-1033.1983.tb07732.x. [DOI] [PubMed] [Google Scholar]
- Ito K., Beckwith J. R. Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane. Cell. 1981 Jul;25(1):143–150. doi: 10.1016/0092-8674(81)90238-5. [DOI] [PubMed] [Google Scholar]
- Ito K., Sato T., Yura T. Synthesis and assembly of the membrane proteins in E. coli. Cell. 1977 Jul;11(3):551–559. doi: 10.1016/0092-8674(77)90073-3. [DOI] [PubMed] [Google Scholar]
- Koshland D., Botstein D. Evidence for posttranslational translocation of beta-lactamase across the bacterial inner membrane. Cell. 1982 Oct;30(3):893–902. doi: 10.1016/0092-8674(82)90294-x. [DOI] [PubMed] [Google Scholar]
- Kumamoto C. A., Oliver D. B., Beckwith J. Signal sequence mutations disrupt feedback between secretion of an exported protein and its synthesis in E. coli. 1984 Apr 26-May 2Nature. 308(5962):863–864. doi: 10.1038/308863a0. [DOI] [PubMed] [Google Scholar]
- Kuritzkes D. R., Zhang X. Y., Lin E. C. Use of phi(glp-lac) in studies of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB). J Bacteriol. 1984 Feb;157(2):591–598. doi: 10.1128/jb.157.2.591-598.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Larson T. J., Ehrmann M., Boos W. Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon. J Biol Chem. 1983 May 10;258(9):5428–5432. [PubMed] [Google Scholar]
- Larson T. J., Schumacher G., Boos W. Identification of the glpT-encoded sn-glycerol-3-phosphate permease of Escherichia coli, an oligomeric integral membrane protein. J Bacteriol. 1982 Dec;152(3):1008–1021. doi: 10.1128/jb.152.3.1008-1021.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ludtke D., Larson T. J., Beck C., Boos W. Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli. Mol Gen Genet. 1982;186(4):540–547. doi: 10.1007/BF00337962. [DOI] [PubMed] [Google Scholar]
- Müller N., Heine H. G., Boos W. Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli. Mol Gen Genet. 1982;185(3):473–480. doi: 10.1007/BF00334143. [DOI] [PubMed] [Google Scholar]
- Neu H. C., Heppel L. A. The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965 Sep;240(9):3685–3692. [PubMed] [Google Scholar]
- Nikaido H., Wu H. C. Amino acid sequence homology among the major outer membrane proteins of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1048–1052. doi: 10.1073/pnas.81.4.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Randall L. L. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell. 1983 May;33(1):231–240. doi: 10.1016/0092-8674(83)90352-5. [DOI] [PubMed] [Google Scholar]
- Reeve J. Use of minicells for bacteriophage-directed polypeptide synthesis. Methods Enzymol. 1979;68:493–503. doi: 10.1016/0076-6879(79)68038-2. [DOI] [PubMed] [Google Scholar]
- Russel M., Model P. Filamentous phage pre-coat is an integral membrane protein: analysis by a new method of membrane preparation. Cell. 1982 Jan;28(1):177–184. doi: 10.1016/0092-8674(82)90387-7. [DOI] [PubMed] [Google Scholar]
- Schryvers A., Weiner J. H. The anaerobic sn-glycerol-3-phosphate dehydrogenase: cloning and expression of the glpA gene of Escherichia coli and identification of the glpA products. Can J Biochem. 1982 Mar;60(3):224–231. doi: 10.1139/o82-027. [DOI] [PubMed] [Google Scholar]
- Shultz J., Silhavy T. J., Berman M. L., Fiil N., Emr S. D. A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery. Cell. 1982 Nov;31(1):227–235. doi: 10.1016/0092-8674(82)90422-6. [DOI] [PubMed] [Google Scholar]
- Silhavy T. J., Benson S. A., Emr S. D. Mechanisms of protein localization. Microbiol Rev. 1983 Sep;47(3):313–344. doi: 10.1128/mr.47.3.313-344.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tommassen J., van Tol H., Lugtenberg B. The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence. EMBO J. 1983;2(8):1275–1279. doi: 10.1002/j.1460-2075.1983.tb01581.x. [DOI] [PMC free article] [PubMed] [Google Scholar]