SORTING MECHANISMS REGULATING MEMBRANE-PROTEIN TRAFFIC IN THE APICAL TRANSCYTOTIC PATHWAY OF POLARIZED MDCK CELLS

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical s urface of polarized MDCK cells has been mapped using morphological tra cers. At 20 degrees C dIgA-pIgR internalize to interconnected groups o f vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) a nd epidermal growth factor receptors (EGFR). Upon transfer to 37 degre es C the endosome vacuoles develop long tubules that give rise to a di stinctive population of 100-nm-diam cup-shaped vesicles containing pIg R. At the same time, the endosome gives rise to multivesicular endosom es (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surfac e where they exocytose. Using Video microscopy and correlative electro n microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contai n TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cu p-shaped vesicles to which the tubules give rise become enriched in dI gA/pIgR. Taken together with the published information available on pI gR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface o f polarized MDCK cells are maintained by a signal-dependent, clathrin- based sorting mechanism that operates along the length of the transcyt otic pathway. We propose that the differential sorting of occupied rec eptors within the MDCK endosome is achieved by this clathrin-based mec hanism continuously retrieving receptors like TR from the pathways tha t deliver pIgR to the apical surface and EGFR to the lysosome.