Distribution and trafficking of MPR300 is normal in the cells accumulated cholesterol in late endocytic compartments: Evidence for early endosome-to-TGN trafficking of MPR300 in NPC fibroblasts

Atsushi Umeda, Hideaki Fujita, Toshio Kuronita, Kaori Hirosako, Masaru Himeno, and Yoshitaka Tanaka

Division of Pharmaceutical Cell Biology, Kyushu University, Fukuoka 812-8582

Corresponding Author: tanakay{at}bioc.phar.kyushu-u.ac.jp

It has previously been reported that an accumulation of cholesterol within late endosomes/lysosomes in Niemann-Pick type C (NPC) disease fibroblasts and cells treated with U18666A that mimic NPC causes impairment of retrograde trafficking of a lysosomal e nzyme receptor, MPR300, from late endosomes to the trans-Golgi network (TGN), leading to retention of the receptor into intralumenal vesicles of late endosomal compartments. In apparent conflict with these results, here we show using three different antib odies against MPR300 that like in normal fibroblasts, MPR300 localizes exclusively in the TGN in NPC fibroblasts as well as in normal fibroblasts treated with U18666A. This TGN localization of MPR300 in NPC fibroblasts can also account for the results tha t several lysosomal properties and functions such as intracellular lysosomal enzymes activity and localization, biosynthesis of cathepsin D, proteins degradation all are exclusively normal in NPC fibroblasts.Å@These results, therefore, suggest that the ac cumulation of cholesterol in late endosomes/lysosomes does not affect the retrieval pathway 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 redistribution of MPR300 to EEA1- and internalized transferrin-positive, but LAMP-2-negative early/recycling endosomes-like vesicles. 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 gives us to provide important insights into the definition of late endosomes as well as the biogenesis of lysosomes.o

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