It has been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) fibroblasts and U18666A-treated cells causes impairment of retrograde trafficking of the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300) from late endosomes to the trans -Golgi network (TGN). In apparent conflict with these results, here we show that as in normal fibroblasts, MPR300 localizes exclusively to the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This localization can explain why several lysosomal properties and functions, such as intracellular lysosomal enzyme activity and localization, the biosynthesis of cathepsin D, and protein degradation, are all normal in NPC fibroblasts. These results, therefore, suggest that the accumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval of MPR300 from endosomes to the TGN. Furthermore, treatment of normal and NPC fibroblasts with chloroquine, which inhibits membrane traffic from early endosomes to the TGN, resulted in a redistribution of MPR300 to EEA1 and internalized transferrin-positive, but LAMP-2-negative, early-recycling endosomes. We propose that in normal and NPC fibroblasts, MPR300 is exclusively targeted from the TGN to early endosomes, from where it rapidly recycles back to the TGN without being delivered to late endosomes. This notion provides important insights into the definition of late endosomes, as well as the biogenesis of lysosomes. Two distinct and related mannose 6-phosphate receptors (MPRs), the cation-dependent mannose 6-phosphate receptor (MPR46) and the cation-independent mannose 6-phosphate/IGF-II receptor (MPR300), are responsible for sorting soluble lysosomal enzymes from the secretory pathway. They have a largely overlapping but apparently distinct affinity for lysosomal enzymes (1, 2). Newly synthesized lysosomal enzymes acquire mannose 6-phosphate residues in the Golgi complex and are recognized in the trans -Golgi network (TGN) by MPRs [reviewed in refs. (3,4)]. The ligand-receptor complex is subsequently packed into clathrin-coated vesicles and targeted to endosomal compartments. The acidic environment of endosomes causes the release of the enzymes from MRPs, after which the lysosomal enzymes are transferred to lysosomes while the MPRs are retrieved to the TGN for further rounds of sorting. Therefore, the MPRs play a key role in the biogenesis and function of lysosomes.Intracellular trafficking of MPRs is mediated by signals present in their cytoplasmic tails, which contain two signals, tyrosine-and dileucine-based sorting signals, as well as by posttranslational modifications, such as the phosphorylation of serine residues and the palmitoylation of cysteine residues (3, 4). Notably, an acidic cluster-dileucine motif that consists of a cluster of acidic amino acid residues followed by dileucine residues is important to the sorting of MPRs from the TGN to endosomes. Recent progress in the study of MPR trafficking from the TGN to e...

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