Mol Gen Genet (1992) 232:40-48 © Springer-Verlag 1992 Topological and functional studies on HlyB of Escherichia coli Ivaylo Gentschev and Werner Goebel Institut fiir Genetik und Mikrobiologie, Universit~itW/irzburg, R6ntgenring 1 l, W-8700 Wiirzburg, FRG ReceivedAugust 6, 1991 Summary. The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of c~-haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, a-helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these trans- membrane segments are relatively large and carry an excess of positively charged amino acids, while the peri- plasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the as- sociation of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyA~ fusion pro- tein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fu- sion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyAs with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane. Key words: E. coli haemolysin - Secretion of haemolysin - Topology and function of HlyB Offprint requests to : W. Goebel Introduction Secretion of Escherichia coli ~-haemolysin into the medi- um requires the HlyB and HlyD proteins encoded by the hly gene cluster (Goebel and Hedgpeth 1982; Wagner et al. 1983; Mackman et al. 1985), and at least one addi- tional outer membrane protein, TolC (Wandersman and Delepelaire 1990). It was suggested that TolC and the integral membrane proteins HlyB and HlyD constitute a specific translocator for haemolysin. HlyC-activated HlyA protein and non-activated HlyA protein are trans- ported by this secretion apparatus with similar efficiency without proteolytic cleavage of an N-terminal signal peptide (Felmlee et al. 1985a). Indeed it was shown that the signal on HlyA that is recognized by the HlyB/HlyD- TolC translocator is located at the C-terminus of HlyA (Mackman et al. 1987) and that the components of the Sec pathway that recognize N-terminal signal peptides may not be required for haemolysin secretion (Gray et al. 1989; Gentschev et al. 1990). The C-terminal HlyA signal sequence has been extensively studied; the data suggest that the last 60 amino acids seem to provide optimal transport efficiency when this peptide is linked to PhoA as reporter protein (Hess et al. 1990), but short- er C-terminal peptides of HlyA were also shown to be secreted by HlyB/HlyD-dependent transport mecha- nisms (Holland et al. 1989; Koronakis et al. 1989; Hess 1989). Furthermore, it was demonstrated that the 60 amino acid C-terminal peptide is the smallest peptide that is still transported by the Hly translocator (Jarchau et al. 1992). Our previous data suggested that the signal peptide of HlyA is recognized by HlyB, since a mutant that lacks functional HlyD can still transport portions of HlyA to the bacterial surface where they can be visual- ized by immunogold-labelled HlyA-specific antibodies (Oropeza-Wekerle et al. 1990). The localization of HlyB and HIyD proteins to the inner membrane was previous- ly reported (Mackman etal. 1985) but substantial amounts of these products, particularly HlyD, have re- peatedly been observed in the outer membrane fractions