Lysosomal biogenesis depends on proper transport of lysosomal enzymes by the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the trans-Golgi network (TGN) to endosomes. Trafficking of the CD-MPR is mediated by sorting signals in its cytoplasmic tail. GGA1 (Golgi-localizing, ␥-ear-containing, ARFbinding protein-1) binds to CD-MPR in the TGN and targets the receptor to clathrin-coated pits for transport from the TGN to endosomes. The motif of the CD-MPR that interacts with GGA1 was

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... wn to be 61 DXXLL 65 . Reports on increased affinity of cargo, when phosphorylated by casein kinase 2 (CK2), to GGAs focused our interest on the effect of the CD-MPR CK2 site on binding to GGA1. Here we demonstrate that Glu 58 and Glu 59 of the CK2 site are essential for high affinity GGA1 binding in vitro, whereas the phosphorylation of Ser 57 of the CD-MPR has no influence on receptor binding to GGA1. Furthermore, the in vivo interaction between GGA1 and CD-MPR was abolished only when all residues involved in GGA1 binding were mutated, namely, Glu 58 , Glu 59 , Asp 61 , Leu 64 , and Leu 65 . In contrast, the binding of adaptor protein-1 (AP-1) to CD-MPR required all the glutamates surrounding the phosphorylation site, namely, Glu 55 , Glu 56 , Glu 58 , and Glu 59 , but like GGA1 binding, was independent of the phosphorylation of Ser 57 . The binding affinity of GGA1 to the CD-MPR was found to be 2.4-fold higher than that of AP-1. This could regulate the binding of the two proteins to the partly overlapping sorting signals, allowing AP-1 binding to the CD-MPR only when GGA1 is released upon autoinhibition by phosphorylation. The cation-dependent mannose 6-phophate receptor (CD-MPR) 1 is a type I integral membrane protein that is involved in the transport of lysosomal hydrolases to the lysosomes (1, 2).