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Role for LAMP-2 in endosomal cholesterol transport.

Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M, Blanz J, Domanskyy M, Breiden B, Brodesser S, Landgrebe J, Sandhoff K, Ikonen E, Saftig P, Eskelinen EL - J. Cell. Mol. Med. (2011)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Kiel, Kiel, Germany.

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Lack of lipid droplets and cholesterol esters in LAMP-1/ LAMP-2 deficient MEFs. (A) Wild-type and LAMP double deficient (LAMP−/−) MEFs were fixed and stained with filipin to detect free cholesterol, or Nile red to detect neutral lipids, as indicated. (B) The percentage of cells with lipid droplets was estimated in wild-type / and LAMP−/− MEFs. (C) Total lipids / of wild-type and LAMP−/− MEFs were analysed using TLC. The quantification of free and esterified cholesterol was done from five experiments and the average and standard deviation are shown. Chol, cholesterol; FA, fatty acids; Cer, ceramide. The asterisks indicate statistical significance: **, P < 0.005; ***, P < 0.001. (D) MEFs were metabolically labelled with [14C] acetate for 48 hrs. Lipids were extracted and same amounts of radioactivity were separated by TLC. The quantification shows mean ± S.E.M. from three experiments. TAG, triacyl glycerol.
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fig01: Lack of lipid droplets and cholesterol esters in LAMP-1/ LAMP-2 deficient MEFs. (A) Wild-type and LAMP double deficient (LAMP−/−) MEFs were fixed and stained with filipin to detect free cholesterol, or Nile red to detect neutral lipids, as indicated. (B) The percentage of cells with lipid droplets was estimated in wild-type / and LAMP−/− MEFs. (C) Total lipids / of wild-type and LAMP−/− MEFs were analysed using TLC. The quantification of free and esterified cholesterol was done from five experiments and the average and standard deviation are shown. Chol, cholesterol; FA, fatty acids; Cer, ceramide. The asterisks indicate statistical significance: **, P < 0.005; ***, P < 0.001. (D) MEFs were metabolically labelled with [14C] acetate for 48 hrs. Lipids were extracted and same amounts of radioactivity were separated by TLC. The quantification shows mean ± S.E.M. from three experiments. TAG, triacyl glycerol.

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 [15] (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.


Role for LAMP-2 in endosomal cholesterol transport.

Schneede A, Schmidt CK, Hölttä-Vuori M, Heeren J, Willenborg M, Blanz J, Domanskyy M, Breiden B, Brodesser S, Landgrebe J, Sandhoff K, Ikonen E, Saftig P, Eskelinen EL - J. Cell. Mol. Med. (2011)

Lack of lipid droplets and cholesterol esters in LAMP-1/ LAMP-2 deficient MEFs. (A) Wild-type and LAMP double deficient (LAMP−/−) MEFs were fixed and stained with filipin to detect free cholesterol, or Nile red to detect neutral lipids, as indicated. (B) The percentage of cells with lipid droplets was estimated in wild-type / and LAMP−/− MEFs. (C) Total lipids / of wild-type and LAMP−/− MEFs were analysed using TLC. The quantification of free and esterified cholesterol was done from five experiments and the average and standard deviation are shown. Chol, cholesterol; FA, fatty acids; Cer, ceramide. The asterisks indicate statistical significance: **, P < 0.005; ***, P < 0.001. (D) MEFs were metabolically labelled with [14C] acetate for 48 hrs. Lipids were extracted and same amounts of radioactivity were separated by TLC. The quantification shows mean ± S.E.M. from three experiments. TAG, triacyl glycerol.
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Related In: Results  -  Collection

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fig01: Lack of lipid droplets and cholesterol esters in LAMP-1/ LAMP-2 deficient MEFs. (A) Wild-type and LAMP double deficient (LAMP−/−) MEFs were fixed and stained with filipin to detect free cholesterol, or Nile red to detect neutral lipids, as indicated. (B) The percentage of cells with lipid droplets was estimated in wild-type / and LAMP−/− MEFs. (C) Total lipids / of wild-type and LAMP−/− MEFs were analysed using TLC. The quantification of free and esterified cholesterol was done from five experiments and the average and standard deviation are shown. Chol, cholesterol; FA, fatty acids; Cer, ceramide. The asterisks indicate statistical significance: **, P < 0.005; ***, P < 0.001. (D) MEFs were metabolically labelled with [14C] acetate for 48 hrs. Lipids were extracted and same amounts of radioactivity were separated by TLC. The quantification shows mean ± S.E.M. from three experiments. TAG, triacyl glycerol.
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 [15] (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.

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, University of Kiel, Kiel, Germany.

Show MeSH