Distribution and trafficking of MPR300 is normal in cells with cholesterol accumulated in late endocytic compartments

Abstract

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...

Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

“…Since U-MPRCs are also detected as punctate signals near the nuclei, it would be quite difficult to detect the early-phase redistribution of CIMPR in the latter cell lines unless the signal was compared with markers for late endosomes, as demonstrated in this study. Very recently, Umeda et al (2003) showed that CIMPR is localized exclusively in the TGN both in control and NPC fibroblasts, and in control fibroblasts treated with U18666A for 24 h. Although the result using U18666A is consistent with ours in that CIMPR is not redistributed into late endosomal compartments after a short duration (24 h) of U18666A treatment, they showed overlaps between the signals for CIMPR and syntaxin 6 or AP-1 by immunofluorescence microscopy, which is in conflict with our results. However, because the study did not discriminate between perinuclear compartments at higher magnifications (e.g., by electron microscopy), it is still possible that CIMPR may actually have been redistributed within the perinuclear region in the fibroblasts at least in the experiment using U18666A as shown in the present study.…”

Phosphatidylinositol 4-Kinaseβ Is Critical for Functional Association of rab11 with the Golgi Complex

“…The nonsignificant effect of the kinase dead mutant of PI4K␤ on VSV-G transport stimulated us to investigate its role in other Golgi-mediated transport processes such as Golgi-toendosome and Golgi-to-endoplasmic reticulum transport. The effect of overexpression of PI4K␤kd on these transport processes was investigated by analyzing the distribution of either the mannose-6-phospate/IGFII receptor (MPR300) and the KDEL-receptor (Bard et al, 2003;Umeda et al, 2003). No alteration in the distribution of both receptors was observed in the presence of the PI4K␤ kinase dead mutant, indicating that the lipid kinase activity is merely involved in the building of the Golgi complex than in transport processes from the Golgi.…”

A Cyclingcis-Golgi Protein Mediates Endosome-to-Golgi Traffic

“…Rather, it seems to be an endosome-to-Golgi route used by a growing list of endogenous proteins that cycle distally out of, and back to, the Golgi apparatus (Ghosh et al, 1998;Mallard et al, 1998;Puri et al, 2002;Medigeshi and Schu, 2003;Umeda et al, 2003;Lin et al, 2004). A defining example is the trafficking itinerary of TGN38/46, which can be contrasted to that of the more classic late endosomal itinerary of the endoprotease furin.…”