Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA.
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 hylA 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.
Jarchau
T
T
Theodor-Boveri-Institut für Biowissenschaften, Würzburg, Germany.
Chakraborty
T
T
Garcia
F
F
Goebel
W
W
D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000378:metabolism*; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments / Q000378:metabolism
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C065231:HlyD protein, E coli; C036507:Hlya protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments