Alteration of the Amino Terminus of the Mature Sequence of a Periplasmic Protein Can Severely Affect Protein Export in Escherichia Coli

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1988 Oct 1

PMID 3051001

Citations 63

Authors

P Li

J Beckwith

H Inouye

Affiliations

Soon will be listed here.

Abstract

Escherichia coli alkaline phosphatase, coded for by the phoA gene, is normally translocated across the cytoplasmic membrane into the periplasm with high efficiency. We have constructed a series of derivatives of the phoA gene that code for a wild-type signal sequence but result in altered amino acid sequences at the amino terminus of the mature alkaline phosphatase. Our results suggest that the presence of two positively charged amino acids very early in the mature sequence interferes significantly with protein export. In one case, phoA2AB, the presence of the sequence Arg-Ile-Arg at the amino terminus of alkaline phosphatase results in a 50-times reduction in the export of the protein. By using oligonucleotide-directed mutagenesis, we have constructed mutant derivatives of phoA2AB that are greatly enhanced for export. In all cases, these derivatives reduce the net positive charge in the region. Our results may explain the failure of E. coli to export a number of proteins coded for by artificial constructs and suggest a way to improve export in these cases.

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References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Heijne G . The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. EMBO J. 1986; 5(11):3021-7. PMC: 1167256. DOI: 10.1002/j.1460-2075.1986.tb04601.x. View

Hoffman C, Wright A . Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985; 82(15):5107-11. PMC: 390508. DOI: 10.1073/pnas.82.15.5107. View

Collier D, Bankaitis V, Weiss J, Bassford Jr P . The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988; 53(2):273-83. DOI: 10.1016/0092-8674(88)90389-3. View

Vlasuk G, Inouye S, Ito H, Itakura K, Inouye M . Effects of the complete removal of basic amino acid residues from the signal peptide on secretion of lipoprotein in Escherichia coli. J Biol Chem. 1983; 258(11):7141-8. View

von Heijne G . Towards a comparative anatomy of N-terminal topogenic protein sequences. J Mol Biol. 1986; 189(1):239-42. DOI: 10.1016/0022-2836(86)90394-3. View

Michaelis S, Inouye H, Oliver D, Beckwith J . Mutations that alter the signal sequence of alkaline phosphatase in Escherichia coli. J Bacteriol. 1983; 154(1):366-74. PMC: 217468. DOI: 10.1128/jb.154.1.366-374.1983. View

Bassford Jr P, Silhavy T, Beckwith J . Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol. 1979; 139(1):19-31. PMC: 216822. DOI: 10.1128/jb.139.1.19-31.1979. View

Liss L, Johnson B, Oliver D . Export defect adjacent to the processing site of staphylococcal nuclease is suppressed by a prlA mutation. J Bacteriol. 1985; 164(2):925-8. PMC: 214342. DOI: 10.1128/jb.164.2.925-928.1985. View

10.

Muller M, Blobel G . Protein export in Escherichia coli requires a soluble activity. Proc Natl Acad Sci U S A. 1984; 81(24):7737-41. PMC: 392227. DOI: 10.1073/pnas.81.24.7737. View

11.

Kuhn A, Wickner W . Conserved residues of the leader peptide are essential for cleavage by leader peptidase. J Biol Chem. 1985; 260(29):15914-8. View

12.

Inouye S, Soberon X, Franceschini T, Nakamura K, Itakura K, Inouye M . Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982; 79(11):3438-41. PMC: 346435. DOI: 10.1073/pnas.79.11.3438. View

13.

Manoil C, Beckwith J . A genetic approach to analyzing membrane protein topology. Science. 1986; 233(4771):1403-8. DOI: 10.1126/science.3529391. View

14.

Michaelis S, Guarente L, Beckwith J . In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli. J Bacteriol. 1983; 154(1):356-65. PMC: 217467. DOI: 10.1128/jb.154.1.356-365.1983. View

15.

Lin J, Kanazawa H, Ozols J, Wu H . An Escherichia coli mutant with an amino acid alteration within the signal sequence of outer membrane prolipoprotein. Proc Natl Acad Sci U S A. 1978; 75(10):4891-5. PMC: 336227. DOI: 10.1073/pnas.75.10.4891. View

16.

Weng Q, Chen L, Tai P . Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles. J Bacteriol. 1988; 170(1):126-31. PMC: 210615. DOI: 10.1128/jb.170.1.126-131.1988. View

17.

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

18.

Emr S, Silhavy T . Mutations affecting localization of an Escherichia coli outer membrane protein, the bacteriophage lambda receptor. J Mol Biol. 1980; 141(1):63-90. DOI: 10.1016/s0022-2836(80)80029-5. View

19.

Russel M, Model P . A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein. Proc Natl Acad Sci U S A. 1981; 78(3):1717-21. PMC: 319204. DOI: 10.1073/pnas.78.3.1717. View

20.

Hall M, Gabay J, Schwartz M . Evidence for a coupling of synthesis and export of an outer membrane protein in Escherichia coli. EMBO J. 1983; 2(1):15-9. PMC: 555079. DOI: 10.1002/j.1460-2075.1983.tb01373.x. View