Fig. 6. Determination of type I membrane topology of TgMIC6, both during its transport and during its storage in the micronemes. (A) The three forms of TgMIC6. The 53, 45 and 35 kDa forms are present in the ER/Golgi, in micronemes and secreted, respectively. The two processing sites are indicated by arrows. (B) Western blot analysis of lysate from wild-type parasites (left panel) after transient permeabilization and proteinase K (0.1 mg/ml and 0.05 mg/ml) treatment either in the presence or absence of detergent (0.2% Triton X-100). The same analysis was repeated using mic1ko mutant parasites (right panel) in which TgMIC6 was retained in the early compartments of the secretory pathway and consequently was not processed at the N-terminus. The western blots were probed with -NterMIC6 antibodies. The same material was analyzed in the lower panels with the -CterMIC6 antibodies, establishing that the tail of TgMIC6 was degraded by proteinase K treatment both in RH and mic1ko strains. (C) A similar experiment was repeated on the recombinant cell line expressing MIC6Ty in mic1ko. In this experiment, TgM2AP, a 43 kDa microneme protein associated with the lumenal domain of TgMIC2, was included as control to demonstrate the integrity of the micronemes.