Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA |
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Authors: | T Jarchau T Chakraborty F Garcia and W Goebel |
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Institution: | (1) Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, 97074 Würzburg, Germany;(2) Present address: Labor f. Klinische Biochemie, Medizinische Universitätsklinik, Josef-Schneider-Str. 2, 97080 Würzburg, Germany;(3) Present address: Abteilung Infektionsimmunologie, Instiut für Medizinische Mikrobiologie, Justus-Liebig Universität Giessen, Frankfurter Str. 107, 35392 Giessen, Germany;(4) Present address: Tropical Disease Research Program, School of Veterinary Medicine, Universidad National, P. O. Box 304-3000, Heredia, Costa Rica |
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Abstract: | Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5 end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA. |
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Keywords: | Escherichia coli hemolysin Transport Signal sequence Peptide transport |
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