Abstract
The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.
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.
- Akita M., Sasaki S., Matsuyama S., Mizushima S. SecA interacts with secretory proteins by recognizing the positive charge at the amino terminus of the signal peptide in Escherichia coli. J Biol Chem. 1990 May 15;265(14):8164–8169. [PubMed] [Google Scholar]
- Akiyama Y., Ito K. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 1987 Nov;6(11):3465–3470. doi: 10.1002/j.1460-2075.1987.tb02670.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Altman E., Emr S. D., Kumamoto C. A. The presence of both the signal sequence and a region of mature LamB protein is required for the interaction of LamB with the export factor SecB. J Biol Chem. 1990 Oct 25;265(30):18154–18160. [PubMed] [Google Scholar]
- Bankaitis V. A., Bassford P. J., Jr Proper interaction between at least two components is required for efficient export of proteins to the Escherichia coli cell envelope. J Bacteriol. 1985 Jan;161(1):169–178. doi: 10.1128/jb.161.1.169-178.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bankaitis V. A., Bassford P. J., Jr The synthesis of export-defective proteins can interfere with normal protein export in Escherichia coli. J Biol Chem. 1984 Oct 10;259(19):12193–12200. [PubMed] [Google Scholar]
- Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
- Bassford P., Beckwith J. Escherichia coli mutants accumulating the precursor of a secreted protein in the cytoplasm. Nature. 1979 Feb 15;277(5697):538–541. doi: 10.1038/277538a0. [DOI] [PubMed] [Google Scholar]
- Bayer M. E., Bayer M. H., Lunn C. A., Pigiet V. Association of thioredoxin with the inner membrane and adhesion sites in Escherichia coli. J Bacteriol. 1987 Jun;169(6):2659–2666. doi: 10.1128/jb.169.6.2659-2666.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beacham I. R. Periplasmic enzymes in gram-negative bacteria. Int J Biochem. 1979;10(11):877–883. doi: 10.1016/0020-711x(79)90117-4. [DOI] [PubMed] [Google Scholar]
- Bedouelle H., Bassford P. J., Jr, Fowler A. V., Zabin I., Beckwith J., Hofnung M. Mutations which alter the function of the signal sequence of the maltose binding protein of Escherichia coli. Nature. 1980 May 8;285(5760):78–81. doi: 10.1038/285078a0. [DOI] [PubMed] [Google Scholar]
- Bieker K. L., Phillips G. J., Silhavy T. J. The sec and prl genes of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):291–310. doi: 10.1007/BF00763169. [DOI] [PubMed] [Google Scholar]
- Blobel G., Dobberstein B. Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma. J Cell Biol. 1975 Dec;67(3):835–851. doi: 10.1083/jcb.67.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bowden G. A., Baneyx F., Georgiou G. Abnormal fractionation of beta-lactamase in Escherichia coli: evidence for an interaction with the inner membrane in the absence of a leader peptide. J Bacteriol. 1992 May;174(10):3407–3410. doi: 10.1128/jb.174.10.3407-3410.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyd D., Beckwith J. Positively charged amino acid residues can act as topogenic determinants in membrane proteins. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9446–9450. doi: 10.1073/pnas.86.23.9446. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
- Brundage L., Fimmel C. J., Mizushima S., Wickner W. SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. J Biol Chem. 1992 Feb 25;267(6):4166–4170. [PubMed] [Google Scholar]
- Brundage L., Hendrick J. P., Schiebel E., Driessen A. J., Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 1990 Aug 24;62(4):649–657. doi: 10.1016/0092-8674(90)90111-q. [DOI] [PubMed] [Google Scholar]
- Calos M. P. DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation. Nature. 1978 Aug 24;274(5673):762–765. doi: 10.1038/274762a0. [DOI] [PubMed] [Google Scholar]
- Chang C. N., Kuang W. J., Chen E. Y. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene. 1986;44(1):121–125. doi: 10.1016/0378-1119(86)90050-8. [DOI] [PubMed] [Google Scholar]
- Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
- Collier D. N., Strobel S. M., Bassford P. J., Jr SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins. J Bacteriol. 1990 Dec;172(12):6875–6884. doi: 10.1128/jb.172.12.6875-6884.1990. [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]
- Derman A. I., Beckwith J. Escherichia coli alkaline phosphatase fails to acquire disulfide bonds when retained in the cytoplasm. J Bacteriol. 1991 Dec;173(23):7719–7722. doi: 10.1128/jb.173.23.7719-7722.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duplay P., Bedouelle H., Fowler A., Zabin I., Saurin W., Hofnung M. Sequences of the malE gene and of its product, the maltose-binding protein of Escherichia coli K12. J Biol Chem. 1984 Aug 25;259(16):10606–10613. [PubMed] [Google Scholar]
- Emr S. D., Bassford P. J., Jr Localization and processing of outer membrane and periplasmic proteins in Escherichia coli strains harboring export-specific suppressor mutations. J Biol Chem. 1982 May 25;257(10):5852–5860. [PubMed] [Google Scholar]
- Emr S. D., Hanley-Way S., Silhavy T. J. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. doi: 10.1016/0092-8674(81)90272-5. [DOI] [PubMed] [Google Scholar]
- Emr S. D., Hedgpeth J., Clément J. M., Silhavy T. J., Hofnung M. Sequence analysis of mutations that prevent export of lambda receptor, an Escherichia coli outer membrane protein. Nature. 1980 May 8;285(5760):82–85. doi: 10.1038/285082a0. [DOI] [PubMed] [Google Scholar]
- Emr S. D., Schwartz M., Silhavy T. J. Mutations altering the cellular localization of the phage lambda receptor, an Escherichia coli outer membrane protein. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5802–5806. doi: 10.1073/pnas.75.12.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Emr S. D., Silhavy T. J. Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980 Jul 25;141(1):63–90. doi: 10.1016/s0022-2836(80)80029-5. [DOI] [PubMed] [Google Scholar]
- Emr S. D., Silhavy T. J. The signal hypothesis in bacteria. Prog Clin Biol Res. 1982;91:3–14. [PubMed] [Google Scholar]
- Fandl J., Tai P. C. Protein translocation in vitro: biochemical characterization of genetically defined translocation components. J Bioenerg Biomembr. 1990 Jun;22(3):369–387. doi: 10.1007/BF00763173. [DOI] [PubMed] [Google Scholar]
- Geider K., Hohmeyer C., Haas R., Meyer T. F. A plasmid cloning system utilizing replication and packaging functions of the filamentous bacteriophage fd. Gene. 1985;33(3):341–349. doi: 10.1016/0378-1119(85)90242-2. [DOI] [PubMed] [Google Scholar]
- Gennity J., Goldstein J., Inouye M. Signal peptide mutants of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):233–269. doi: 10.1007/BF00763167. [DOI] [PubMed] [Google Scholar]
- Hardy S. J., Randall L. L. A kinetic partitioning model of selective binding of nonnative proteins by the bacterial chaperone SecB. Science. 1991 Jan 25;251(4992):439–443. doi: 10.1126/science.1989077. [DOI] [PubMed] [Google Scholar]
- He M., Wilde A., Kaderbhai M. A. A simple single-step procedure for small-scale preparation of Escherichia coli plasmids. Nucleic Acids Res. 1990 Mar 25;18(6):1660–1660. doi: 10.1093/nar/18.6.1660. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoyt D. W., Gierasch L. M. A peptide corresponding to an export-defective mutant OmpA signal sequence with asparagine in the hydrophobic core is unable to insert into model membranes. J Biol Chem. 1991 Aug 5;266(22):14406–14412. [PubMed] [Google Scholar]
- Hoyt D. W., Gierasch L. M. Hydrophobic content and lipid interactions of wild-type and mutant OmpA signal peptides correlate with their in vivo function. Biochemistry. 1991 Oct 22;30(42):10155–10163. doi: 10.1021/bi00106a012. [DOI] [PubMed] [Google Scholar]
- Inouye H., Michaelis S., Wright A., Beckwith J. Cloning and restriction mapping of the alkaline phosphatase structural gene (phoA) of Escherichia coli and generation of deletion mutants in vitro. J Bacteriol. 1981 May;146(2):668–675. doi: 10.1128/jb.146.2.668-675.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito K., Wittekind M., Nomura M., Shiba K., Yura T., Miura A., Nashimoto H. A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell. 1983 Mar;32(3):789–797. doi: 10.1016/0092-8674(83)90065-x. [DOI] [PubMed] [Google Scholar]
- Jacobson G. R., Takacs B. J., Rosenbusch J. P. Properties of a major protein released from Escherichia coli by osmotic shock. Biochemistry. 1976 Jun 1;15(11):2297–2303. doi: 10.1021/bi00656a008. [DOI] [PubMed] [Google Scholar]
- Jones J. D., McKnight C. J., Gierasch L. M. Biophysical studies of signal peptides: implications for signal sequence functions and the involvement of lipid in protein export. J Bioenerg Biomembr. 1990 Jun;22(3):213–232. doi: 10.1007/BF00763166. [DOI] [PubMed] [Google Scholar]
- Kumamoto C. A., Beckwith J. Evidence for specificity at an early step in protein export in Escherichia coli. J Bacteriol. 1985 Jul;163(1):267–274. doi: 10.1128/jb.163.1.267-274.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumamoto C. A. SecB protein: a cytosolic export factor that associates with nascent exported proteins. J Bioenerg Biomembr. 1990 Jun;22(3):337–351. doi: 10.1007/BF00763171. [DOI] [PubMed] [Google Scholar]
- Laminet A. A., Plückthun A. The precursor of beta-lactamase: purification, properties and folding kinetics. EMBO J. 1989 May;8(5):1469–1477. doi: 10.1002/j.1460-2075.1989.tb03530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li P., Beckwith J., Inouye H. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7685–7689. doi: 10.1073/pnas.85.20.7685. [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]
- Lill R., Dowhan W., Wickner W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 1990 Jan 26;60(2):271–280. doi: 10.1016/0092-8674(90)90742-w. [DOI] [PubMed] [Google Scholar]
- Liu G., Topping T. B., Randall L. L. Physiological role during export for the retardation of folding by the leader peptide of maltose-binding protein. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9213–9217. doi: 10.1073/pnas.86.23.9213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michaelis S., Hunt J. F., Beckwith J. Effects of signal sequence mutations on the kinetics of alkaline phosphatase export to the periplasm in Escherichia coli. J Bacteriol. 1986 Jul;167(1):160–167. doi: 10.1128/jb.167.1.160-167.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milstein C., Brownlee G. G., Harrison T. M., Mathews M. B. A possible precursor of immunoglobulin light chains. Nat New Biol. 1972 Sep 27;239(91):117–120. doi: 10.1038/newbio239117a0. [DOI] [PubMed] [Google Scholar]
- Nishiyama K., Kabuyama Y., Akimaru J., Matsuyama S., Tokuda H., Mizushima S. SecY is an indispensable component of the protein secretory machinery of Escherichia coli. Biochim Biophys Acta. 1991 May 31;1065(1):89–97. doi: 10.1016/0005-2736(91)90015-z. [DOI] [PubMed] [Google Scholar]
- Nygren H., Rozell B., Holmgren A., Hansson H. A. Immunoelectron microscopic localization of glutaredoxin and thioredoxin in Escherichia coli cells. FEBS Lett. 1981 Oct 12;133(1):145–150. doi: 10.1016/0014-5793(81)80492-9. [DOI] [PubMed] [Google Scholar]
- Oliver D. B., Cabelli R. J., Dolan K. M., Jarosik G. P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8227–8231. doi: 10.1073/pnas.87.21.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Park S., Liu G., Topping T. B., Cover W. H., Randall L. L. Modulation of folding pathways of exported proteins by the leader sequence. Science. 1988 Feb 26;239(4843):1033–1035. doi: 10.1126/science.3278378. [DOI] [PubMed] [Google Scholar]
- Puziss J. W., Strobel S. M., Bassford P. J., Jr Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol. 1992 Jan;174(1):92–101. doi: 10.1128/jb.174.1.92-101.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Randall L. L., Hardy S. J., Thom J. R. Export of protein: a biochemical view. Annu Rev Microbiol. 1987;41:507–541. doi: 10.1146/annurev.mi.41.100187.002451. [DOI] [PubMed] [Google Scholar]
- Randall L. L., Topping T. B., Hardy S. J. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. doi: 10.1126/science.2188362. [DOI] [PubMed] [Google Scholar]
- Rasmussen B. A., MacGregor C. H., Ray P. H., Bassford P. J., Jr In vivo and in vitro synthesis of Escherichia coli maltose-binding protein under regulatory control of the lacUV5 promoter-operator. J Bacteriol. 1985 Nov;164(2):665–673. doi: 10.1128/jb.164.2.665-673.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ross G. W., O'Callaghan C. H. Beta-lactamase assays. Methods Enzymol. 1975;43:69–85. doi: 10.1016/0076-6879(75)43081-6. [DOI] [PubMed] [Google Scholar]
- Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
- Ryan J. P., Bassford P. J., Jr Post-translational export of maltose-binding protein in Escherichia coli strains harboring malE signal sequence mutations and either prl+ or prl suppressor alleles. J Biol Chem. 1985 Nov 25;260(27):14832–14837. [PubMed] [Google Scholar]
- San Millan J. L., Boyd D., Dalbey R., Wickner W., Beckwith J. Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase. J Bacteriol. 1989 Oct;171(10):5536–5541. doi: 10.1128/jb.171.10.5536-5541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
- Shuman H. A. Active transport of maltose in Escherichia coli K12. Role of the periplasmic maltose-binding protein and evidence for a substrate recognition site in the cytoplasmic membrane. J Biol Chem. 1982 May 25;257(10):5455–5461. [PubMed] [Google Scholar]
- Simon S. M., Blobel G. Signal peptides open protein-conducting channels in E. coli. Cell. 1992 May 15;69(4):677–684. doi: 10.1016/0092-8674(92)90231-z. [DOI] [PubMed] [Google Scholar]
- Strauch K. L., Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576–1580. doi: 10.1073/pnas.85.5.1576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tommassen J. Fallacies of E. coli cell fractionations and consequences thereof for protein export models. Microb Pathog. 1986 Jun;1(3):225–228. doi: 10.1016/0882-4010(86)90046-x. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
- Watanabe M., Blobel G. SecB functions as a cytosolic signal recognition factor for protein export in E. coli. Cell. 1989 Aug 25;58(4):695–705. doi: 10.1016/0092-8674(89)90104-9. [DOI] [PubMed] [Google Scholar]
- Weiss J. B., Bassford P. J., Jr The folding properties of the Escherichia coli maltose-binding protein influence its interaction with SecB in vitro. J Bacteriol. 1990 Jun;172(6):3023–3029. doi: 10.1128/jb.172.6.3023-3029.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss J. B., MacGregor C. H., Collier D. N., Fikes J. D., Ray P. H., Bassford P. J., Jr Factors influencing the in vitro translocation of the Escherichia coli maltose-binding protein. J Biol Chem. 1989 Feb 15;264(5):3021–3027. [PubMed] [Google Scholar]
- de Cock H., Tommassen J. SecB-binding does not maintain the translocation-competent state of prePhoE. Mol Microbiol. 1992 Mar;6(5):599–604. doi: 10.1111/j.1365-2958.1992.tb01506.x. [DOI] [PubMed] [Google Scholar]