A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium.

Escherichia coli haemolysin (HlyA), a 107K (K = 10(3) Mr) protein, is secreted to the medium in an hlyB, hlyD-dependent process. Secretion, however, depends on neither an N-terminal signal sequence nor on SecA, which is part of the normal cellular export machinery for periplasmic and outer membrane proteins. In contrast, HlyA contains a novel C-terminal secretion signal encompassing the last 27 amino acids and possibly some additional residues immediately upstream. This region is characterized by a 16 residue 'aspartic acid box' composed largely of small amino acids which we propose constitutes an important element in recognition of the membrane translocation complex constituted by HlyB and HlyD. This feature is also found at the C-terminus of the adenyl cyclase and leukotoxin A molecules and resembles a recently identified eukaryotic C-terminal signal for targeting to glycosomes. A domain of the HlyB component of the haemolysin transport system is also similar to a domain widely distributed in nature, apparently acting as an ATP-dependent transport protein for a wide variety of molecules. Secretion of haemolysin, however, is the first example of a protein translocation system involving an HlyB-like molecule. This suggests that a major role of HlyB or at least its C-terminal domain is the coupling of energy to translocation of the haemolysin. It is more likely therefore that HlyD is more involved in the actual translocation through the membrane. On the basis of genetical and biochemical studies we propose that the haemolysin is translocated directly to the medium bypassing the periplasm. We further propose that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex (conceivably involving other E. coli membrane proteins) must somehow straddle the inner and outer membranes. Finally, the HlyA C-terminal domain has been successfully used to promote the secretion to the medium of a number of heterologous polypeptides, in an HlyB,D-dependent manner.