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

HlyD has a single transmembrane domain (residues 59-80) and a large peripla smic domain, and is essential for the secretion of haemolysin from Escheric hia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protei n in the presence and absence of other putative components of the transloca tor, HlyB and TolC. HlyD is normally highly stable but in the absence of To lC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37 degrees C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractiona te with the inner membrane, indicating in this case limited cleavage at spe cific sites. However, the effect of removing both HlyB and TolC is not addi tive. On the contrary, in the absence of both HlyB and TorC the half-life o f HIyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence o f both HlyB and TolC. This suggests that the presence of HlyB induces a str uctural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction be tween HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HIyD assembles into the inner membrane displaying t he same stability with and without HlyB as wild type HlyD does. This N-term inal. region therefore appears to play no role in stable localisation but i s involved in secretion, since HIyD22 is completely secretion defective. Mo dification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active h aemolysin is limited to a brief period during mid to late exponential phase . In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the t ranslocator is not the limiting factor for growth phase-dependent secretion .