Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator.

摘要: HlyD has a single transmembrane domain (residues 59-80) and large periplasmic domain, is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, protein was localised to cell envelope by immunofluorescence cytoplasmic membrane sucrose gradient analysis. We have examined stability this in presence absence other putative components translocator, HlyB TolC. normally highly stable but TolC, steady-state level greatly reduced half-life at 37° C 36 min. In HlyB, also unstable specific degradation products are detected, which co-fractionate with inner membrane, indicating case limited cleavage sites. However, effect removing both TolC not additive. On contrary, approximately 110 This result shows that removal renders more than it suggests induces structural change HlyD. addition, itself appears be less These results consistent interaction between HlyD/TolC HlyB/HlyD. A derivative HlyD22, lacking 40 N-terminal residues assembles into displaying same without as wild type does. region therefore play no role localisation involved secretion, since HlyD22 completely defective. Modification C-terminus on hand destabilised molecule detectable envelope. Secretion active brief period during mid late exponential phase. contrast, apparently synthesised constitutively throughout growth phase, demonstrating production component translocator limiting factor phase-dependent secretion.