BASOLATERAL SORTING OF THE CATION-DEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR IN MADIN-DARBY CANINE KIDNEY-CELLS - IDENTIFICATION OF A BASOLATERAL DETERMINANT UNRELATED TO CLATHRIN-COATED PIT LOCALIZATION SIGNALS

In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membra ne proteins in the trans-Golgi network for basolateral delivery depend s on the presence of cytoplasmic determinants that are related or unre lated to clathrin-coated pit localization signals, Whether these signa ls mediate basolateral protein sorting through common or distinct path ways is unknown, The cytoplasmic domain of the cation-dependent mannos e B-phosphate receptor (CD-MPR) contains clathrin-coated pit localizat ion signals that are necessary for endocytosis and lysosomal enzyme ta rgeting, In this study, we have addressed the function of these signal s in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the trans membrane and cytoplasmic mic domains of the CD-MPR was stably expresse d in MDCK cells, This chimera (HCD) is able to interact with the AP-1 Golgi specific assembly proteins and is detected on the basolateral pl asma membrane of MDCK cells where it is endocytosed, Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate, A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail i s also found on the basolateral membrane but is unable to interact wit h the AP-1 assembly proteins, However, the overexpression of this muta nt results in partial apical delivery, It is concluded, therefore, tha t the basolateral transport of this chimera requires a saturable sorti ng machinery distinct from AP-1.