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 proteins and clathrin. The mannose 6-phosphate receptors (MPHs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly, 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-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes 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 the CD-PDR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.
Mauxion
F
F
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Le Borgne
R
R
Munier-Lehmann
H
H
Hoflack
B
B
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001483:Base Sequence; D047390:Casein Kinase II; D002402:Cathepsin D / Q000378:metabolism; D002451:Cell Compartmentation; D002478:Cells, Cultured; D018699:Coated Vesicles / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D017527:Receptor, IGF Type 2; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D002402:Cathepsin D