Parsot, unpublished observation). The secreton structure is composed of three different parts: (a) an external nee- dle, (b) a neck, and (c) a large proximal bulb. In our im- ages, it is difficult to discern the two bacterial membranes and thus to judge the localization of the bulb. Nevertheless the length of the neck domain is sufficient to traverse both membranes, implying that the bulb lies within bacterial cy- tosol. This putative cytoplasmic part was not recovered by Kubori et al. (1998) in secreton preparations, which were purified using a protocol derived from that for isolation of flagellar basal bodies (Aizawa et al., 1985). A cytoplasmic structure (other than the C-ring) is known to be associated with the bacterial flagellum and thought to contain the cy- toplasmic F1-type ATPase FliI as well as several other components. This structure is not recovered in purified basal body preparations and has never been visualized (MacNab, 1996; Minamino and MacNab, 1999). Inactivation of spa47, the homologue of the flagellar biosynthesis gene fliI, abolishes secretion. However, so far, it was unclear whether this ATPase was required for assembly and/or activity of the secretion machinery (Drey- fus et al., 1993). We showed that type III secretons were assembled by S. flexneri before host contact and that their structure did not grossly change when secretion was acti- vated (in the presence of Congo red or in the ipaD or ipaB mutants). We also showed that secretion required energy and temperatures above 308C (Fig. 3, B and D). Low tem- peratures did not disassemble secretons (not shown). Thus ATP hydrolysis and conformational change(s) are likely to be required for Ipa secretion, as for flagellin export (Vo- gler et al., 1991). We propose that Spa47 is the azide-sensi- tive element of the secretion machine. Mechanism for Assembly of the Translocator Intimate contact between bacteria and RBCs is essential for hemolysis. Indeed, secretion is necessary but not suffi- cient for hemolysis since inducing protein secretion from AfaE-expressing bacteria bound to RBCs did not provoke hemolysis in the absence of centrifugation. The increased hemolysis which is seen after centrifugation could be due to the greater surface contact which either activates more secretons to secrete and/or allows Ipas to reach the mem- brane still in active form after release. Both notions are supported by the sharp threshold of g forces required to obtain lysis and by the sialic acid removal experiment (Ta- ble I). Wild-type Shigella do not need centrifugation or the AfaE adhesin to enter HeLa cells, these agents simply make the process more efficient (Clerc and Sansonetti, 1987). The shortened glycocalyx, especially on the basal surface of transformed epithelial cells, might explain why AfaE can increase the efficiency of HeLa cell entry but not of hemolysis. Electron microscopy images indicate that the minimal distance required for lysis is z100 nm; the secreton needle is 60 nm long. Yet, contact with the needle alone is unable to cause lysis as (a) the diameter of the pore (25 Å) is smaller than that of the needle (80–100 Å), and (b) ipaB, ipaC and ipaD mutants display normal secretons yet no hemolysis. Two hypotheses have been put forward for how Ipas as- sociate with host cells (reviewed in Hueck, 1998). First, Ipa proteins could be presented at the bacterial surface and in- serted from there upon contact. This corresponds to a two- step model. Second, contact may induce secretion and the newly secreted Ipas may insert into the target membrane at this stage. This is a one-step model. We detected only minute amounts of Ipas at the bacterial surface by im- munoelectron microscopy and these Ipa proteins never colocalized with secretons revealed by negative staining (Gounon, P., and A. Blocker, unpublished observations). In addition, hemolysis was insensitive to protease pretreat- ment of bacteria, suggesting that the biologically active Ipa proteins are not at the bacterial surface before contact. This is not in favor of a two-step mechanism for Ipa pro- tein transfer into the target membrane. On the other hand, the temperature and energetic correlation between secre- tion and lysis and the additive inhibition of hemolysis by azide and proteinase K (Fig. 3) support a process where secretion or surface presentation upon contact is kineti- cally coupled to insertion. How might secretion and membrane association of the pore forming Ipas be coupled? The approximate diameter of the canal known to exist in the bacterial flagellum is 20– 30 Å (Morgan et al., 1995). As the secreton structure is evolutionarily derived from flagella there could exist a ca- nal of similar diameter through the needle. The semi- folded state from which IpaB and IpaC would necessarily exit the secreton needle (given the small diameter of the putative canal) might be required for their membrane as- sociation. As the role of the Spa47 ATPase is probably to push the secreted proteins into the secreton canal, ATP hydrolysis by this protein could also indirectly serve to al- low membrane insertion. Accordingly, the needle may be required both as a tactile sensor for activation of the secre- ton and to present the Ipas directly at the host membrane surface in a sufficiently unfolded state to allow their inser- tion. What we can not yet assess is whether the semifolded IpaB/IpaC complex enters the membrane immediately upon exiting the secreton or whether there is a short-lived intermediate in a different conformation which travels ,100 Å through the medium to reach the membrane. Nev- ertheless, as 25 Å is the functional inner diameter of the IpaB/IpaC pore we find in RBCs, we propose that the IpaB/IpaC complex fits around the end of the putative ca- nal within the needle. This linkage is likely to be dynamic in time and/or fragile because otherwise osmoprotection would be difficult to rationalize and bacteria would copu- rify with floated RBC membranes after hemolysis. The 25 Å IpaB/IpaC pore would then let the other Ipa proteins destined to be translocated through in the semi-folded state generated by Spa47-powered extrusion from a simi- larly seized secreton canal. This work is dedicated to the memory of Thomas E. Kreis. We are grate- ful to Jacomine Krinjse-Locker for instructions on the use of neuramini- dase and to Michel Chignard and Jean-Claude Manuguerra for human blood and HA assistance. A. Blocker wishes to thank in particular Phil- ippe Clerc for his recollections of 15-yr-old unpublished data on the con- tact hemolytic activity of S. flexneri. She acknowledges Chantal LeBou- genec, Tony Pugsley, and Elie Dassa for local advice and reagents and Lina Bernardini, Guy Cornelis, Mike Donnenberg, Bob MacNab, Ira Mellman, and Dennis Thomas for inspiring discussions, communication of results before publication, and/or critical reading of the manuscript. Fi- nally, she is indebted to Gisou van der Goot, Gareth Griffiths, David Holden, and Paul Lazarow for their supportive friendship. A. Blocker and K. Niebuhr were supported by National Institutes of The Journal of Cell Biology, Volume 147, 1999 692