Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes.
We have studied the C-terminal signal which directs the complete export of the 1024-amino-acid hemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Isolation and sequencing of homologous hlyA genes from the related bacteria Proteus vulgaris and Morganella morganii revealed high primary sequence divergence in the three HlyA C-termini and highlighted within the extreme terminal 53 amino acids the conservation of three contiguous sequences, a potential 18-amino-acid amphiphilic alpha-helix, a cluster of charged residues, and a weakly hydrophobic terminal sequence rich in hydroxylated residues. Fusion of the C-terminal 53 amino acid sequence to non-exported truncated Hly A directed wild-type export but export was radically reduced following independent disruption or progressive truncation of the three C-terminal features by in-frame deletion and the introduction of translation stop codons within the 3' hlyA sequence. The data indicate that the HlyA C-terminal export signal comprises multiple components and suggest possible analogies with the mitochondrial import signal. Hemolysis assays and immunoblotting confirmed the intracellular accumulation of non-exported HlyA proteins and supported the view that export proceeds without a periplasmic intermediate. Comparison of cytoplasmic and extracellular forms of an independently exported extreme C-terminal 194 residue peptide showed that the signal was not removed during export.
Koronakis
V
V
Cambridge University Department of Pathology, UK.
Koronakis
E
E
Hughes
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011511:Proteus / Q000235:genetics; D015398:Signal Transduction
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D021382:Protein Sorting Signals