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Secretion of Ricinus communis glyceraldehyde‐3‐phosphate dehydrogenase by Escherichia coli
Author(s) -
Hekman W. E.,
Dennis D. T.,
Miernyk J. A.
Publication year - 1990
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1990.tb00715.x
Subject(s) - biology , signal peptide , biochemistry , escherichia coli , bacterial outer membrane , signal peptidase , complementary dna , fusion protein , glyceraldehyde 3 phosphate dehydrogenase , microbiology and biotechnology , dehydrogenase , peptide sequence , enzyme , recombinant dna , gene
Summary A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer‐membrane protein A to the 5′‐end of a glyceraldehyde‐3‐phosphate dehydrogenase cDNA from Ricinus communis. When expressed in E. coli , the fusion protein was secreted by the normal membrane‐potential‐dependent pathway. Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol. Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane. The protein could not be released from the membrane by washing with alkaline sodium carbonate. Radioactivity from [U‐ 14 C]‐palmitate was incorporated into the heterologous protein. These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid‐modification site, and the combination of a signal sequence plus a lipid‐modification sequence results in specific targeting to the bacterial outer membrane.