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 c ells expressing wild-type and mutant receptors were analyzed by metabo lic labeling with [H-3]palmitate, immunoprecipitation, and SDS-PAGE, B oth Cys(30) and Cys(34) were found to be sites of palmitoylation and t ogether they accounted for the total palmitoylation of the receptor. T he palmitate rapidly turned over with a half-life of similar to 2 h co mpared to a half-life of greater than 40 h for the protein. Mutation o f 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 al anines impaired palmitoylation of Cys(30) and Cys(34) and resulted in abnormal receptor trafficking to lysosomes and loss of cathepsin D sor ting. These data suggest that palmitoylation of Cys(30) and Cys(34) le ads to anchoring of this region of the cytoplasmic tail to the lipid b ilayer. Anchoring via Cys(34) is essential for the normal trafficking and lysosomal enzyme sorting function of the receptor.