Cysteine(34) of the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor is reversibly palmitoylated and required for normal trafficking and lysosomal enzyme sorting

We have examined whether the two cysteine residues (Cys(30) and Cys(34)) in the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor are palmitoylated via thioesters and whether these residues influence the biologic function of the receptor. To do this, mouse L cells expressing wild-type and mutant receptors were analyzed by metabolic labeling with [H-3]palmitate, immunoprecipitation, and SDS-PAGE, Both Cys(30) and Cys(34) were found to be sites of palmitoylation and together they accounted for the total palmitoylation of the receptor. The palmitate rapidly turned over with a half-life of similar to 2 h compared to a half-life of greater than 40 h for the protein. Mutation of Cys(34) to Ala resulted in the gradual accumulation of the receptor in dense lysosomes and the total loss of cathepsin D sorting function in the Golgi. A Cys(30) to Ala mutation had no biologic consequences, showing the importance of Cys(34). Mutation of amino acids 35-39 to alanines impaired palmitoylation of Cys(30) and Cys(34) and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sorting. These data suggest that palmitoylation of Cys(30) and Cys(34) leads to anchoring of this region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys(34) is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.