A CASEIN KINASE-II PHOSPHORYLATION SITE IN THE CYTOPLASMIC DOMAIN OF THE CATION-DEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR DETERMINES THE HIGH-AFFINITY INTERACTION OF THE AP-1 GOLGI ASSEMBLY PROTEINS WITH MEMBRANES

The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly pr oteins and clathrin. The mannose 6-phosphate receptors (MPRs) are two major transmembrane proteins segregated into these transport vesicles, Together with the GTPase ARF-1, these cargo proteins are essential co mponents for the efficient translocation of the cytosolic AP-1 onto me mbranes of the trans-Golgi network, the first step of clathrin coat as sembly. MPR-negative fibroblasts have a low capacity of recruiting AP- 1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-depende nt mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is es sential for these interactions. Thus, the affinity of AP-1 for membran es and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires two distinct determinants at the carboxyl terminus of the CD-MPR cytoplasmic domain that are di fferent from tyrosine-based endocytosis motifs, The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affi nity binding of AP-1 and therefore probably acts as a dominant determi nant controlling CD-MPR sorting in the trans Golgi network. The second is the adjacent di-leucine motif (RLLPM), which, by itself, is not cr itical for AP-1 binding, but is absolutely required for a downstream s orting event.