APICAL AND BASOLATERAL COATED PITS OF MDCK CELLS DIFFER IN THEIR RATES OF MATURATION INTO COATED VESICLES, BUT NOT IN THE ABILITY TO DISTINGUISH BETWEEN MUTANT HEMAGGLUTININ PROTEINS WITH DIFFERENT INTERNALIZATION SIGNALS

In polarized epithelial MDCK cells, all known endogenous endocytic rec eptors are found on the basolateral domain. The influenza virus hemagg lutinin (HA) which is normally sorted to the apical plasma membrane, c an be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomp lete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the a pical and basolateral surfaces of MDCK cells. The rates of internaliza tion of HA-Y543 and HA-Y543,R546 from the basolateral surface of polar ized MDCK cells resembled those in nonpolarized cells, whereas their r ates of internalization from the apical cell surface were fivefold slo wer. However, HA-Y543,R546 was internalized approximately threefold fa ster than HA-Y543 at both membrane domains, indicating that apical end ocytic pits in polarized MDCK cells retained the ability to discrimina te between different internalization signals. Slower internalization f rom the apical surface could not be explained by a limiting number of coated pits; apical membrane contained 0.7 as many coated pits per cel l cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a pe rcentage not significantly different from that observed in apical coat ed pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apica l coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than wer e seen at the basolateral. These results suggest that the slower inter nalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signa ls.