BASOLATERAL TO APICAL TRANSCYTOSIS IN POLARIZED CELLS IS INDIRECT ANDINVOLVES BFA AND TRIMERIC-G PROTEIN SENSITIVE PASSAGE THROUGH THE APICAL ENDOSOME

We have used temperature and nocodazole blocks in an in vivo basolater al to apical transcytosis assay to dissociate the early transcytotic s teps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([ Fab]2 fragment of IgG against the receptor's ectodomain) at 17-degrees -C but do not deliver it to the apical PM. Instead, the ligand accumul ates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule-requiring translocation step. We have characterized this compartment and show that it is distinct f rom basolateral transferrin recycling endosomes, basolateral early end osomes or late endosomes or lysosomes. The apical transcytotic compart ment colocalizes with the compartment containing apically recycling me mbrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid ph ase marker (BSA). This compartment is an intermediate station of the o verall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17-degrees- C are subsequently incubated at 37-degrees-C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of tri meric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gsalpha, to cells preloaded with transcyt otic ligand at 17-degrees-C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and functi on of the apical endosome are not affected since neither BFA nor chole ra toxin inhibit the recycling of apically internalized membrane marke rs (ricin and pIgA-R ligand) from the same compartment. The data sugge st that transcytosis connects the ''membrane-sorting'' sub-domain of t he basolateral endosome with a homologous sub-domain of the apical end osome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, disti nct from that used for recycling of apically internalized proteins (ri cin or pIgA-R).