Role for LAMP-2 in endosomal cholesterol transport.
Bottom Line: These results point to a critical role for LAMP-2 in endosomal/lysosomal cholesterol export.The LAMP-2 luminal domain, the membrane-proximal half in particular, was necessary and sufficient for the rescue effect.Taken together, our results suggest that LAMP-2, its luminal domain in particular, plays a critical role in endosomal cholesterol transport and that this is distinct from the chaperone-mediated autophagy function of LAMP-2.
Affiliation: Institute of Biochemistry, University of Kiel, Kiel, Germany.Show MeSH
Mentions: We reported earlier that LAMP−/− MEFs show increased total cholesterol levels, accumulate free cholesterol in late endo-somes/lysosomes, and contain reduced amounts of lipid droplets  (Fig. 1A). We confirmed the latter finding by quantitative analysis which showed that lipid droplets were present approximately 3.5 times more often in wild-type MEFs than in LAMP−/− MEFs (Fig. 1B). Similar results were obtained using Sudan III and Bodipy 493/503 staining (not shown). We also used TLC to analyse the proportions of free and esterified cholesterol. As expected, we observed that in LAMP−/− MEFs, the percentage of free cholesterol was increased to 67.3% when compared to the 46.1% in wild-type MEFs (Fig. 1C). These findings suggest that cholesterol esterification is defective in LAMP−/− MEFs. In order to test this, we labelled the cells metabolically with [14C] acetate to follow the esterification of newly-made cholesterol. The results showed a substantial defect in the esterification of cholesterol in LAMP−/− MEFs (Fig. 1D). The metabolic labelling also suggested that LAMP deficient MEFs synthesized less triacylglycerol than wild-type MEFs (Fig. 1D), which is consistent with the decreased amount of lipid droplets.
Affiliation: Institute of Biochemistry, University of Kiel, Kiel, Germany.