Limits...
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.

Show MeSH
Expression of LAMP-2influ-ences cholesterol levels. (A) LAMP-2 deficient liver accumulates cholesterol. Left panel: Total cholesterol was estimated from five wild-type, three LAMP-1–/–, and three LAMP-2−/− livers. The error bars represent standard deviation. The difference between the control and / LAMP-2−/− is statistically significant (P < 0.001). Middle and right panels: Filipin staining of liver sections from six-month-old wild-type / and LAMP-2−/− mice. (B) Gel filtration analysis of plasma lipoprotein / profiles of LAMP-2−/− mice and heterozygote littermate controls (please note that lamp-2 gene is located in the Y chromosome). Total cholesterol levels in the fractions are shown. Plasma samples from four mice were pooled for each group. Note that both female and male / LAMP-2−/− mice show elevated LDL cholesterol levels. (C, D) Overexpression of LAMP-2A retards the U18666A induced cholesterol accumulation. (C) HeLa cells were transfected with mouse LAMP-2A and grown for 29 hrs. The cells were then treated with increasing concentrations of U18666A for 15 hrs, and processed for filipin staining and immunolabelling with antimouse LAMP-2. Asterisks indicate the LAMP-2A overexpressing cells in the left panels. (D) The percentage of cells with late endosomal/lysosomal cholesterol accumulation was estimated. None, HeLa cells with no overexpression; mLA-2A, HeLa cells overexpressing mouse LAMP-2A. The results are the mean and standard deviation from one experiment with two parallel samples. The experiment was repeated with similar results.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3822795&req=5

fig04: Expression of LAMP-2influ-ences cholesterol levels. (A) LAMP-2 deficient liver accumulates cholesterol. Left panel: Total cholesterol was estimated from five wild-type, three LAMP-1–/–, and three LAMP-2−/− livers. The error bars represent standard deviation. The difference between the control and / LAMP-2−/− is statistically significant (P < 0.001). Middle and right panels: Filipin staining of liver sections from six-month-old wild-type / and LAMP-2−/− mice. (B) Gel filtration analysis of plasma lipoprotein / profiles of LAMP-2−/− mice and heterozygote littermate controls (please note that lamp-2 gene is located in the Y chromosome). Total cholesterol levels in the fractions are shown. Plasma samples from four mice were pooled for each group. Note that both female and male / LAMP-2−/− mice show elevated LDL cholesterol levels. (C, D) Overexpression of LAMP-2A retards the U18666A induced cholesterol accumulation. (C) HeLa cells were transfected with mouse LAMP-2A and grown for 29 hrs. The cells were then treated with increasing concentrations of U18666A for 15 hrs, and processed for filipin staining and immunolabelling with antimouse LAMP-2. Asterisks indicate the LAMP-2A overexpressing cells in the left panels. (D) The percentage of cells with late endosomal/lysosomal cholesterol accumulation was estimated. None, HeLa cells with no overexpression; mLA-2A, HeLa cells overexpressing mouse LAMP-2A. The results are the mean and standard deviation from one experiment with two parallel samples. The experiment was repeated with similar results.

Mentions: We showed previously that, in LAMP−/− MEFs, the filipin staining (detecting free cholesterol) was increased and the level of total cholesterol was approximately twofold higher than in wild-type MEFs [15]. In LAMP-2 single deficient MEFs, the filipin staining intensity and cholesterol level were intermediate to wild-type and LAMP−/− MEFs. In order to test whether this phenotype would be observed in LAMP-2 deficient tissue, we investigated cholesterol levels in the liver of mice deficient in LAMP-1 or LAMP-2. Quantitative cholesterol assay showed that the level of total cholesterol was approximately twofold in the LAMP-2 deficient liver compared to the wild-type liver, whereas the level of LAMP-1 deficient liver was not different from the wild-type (Fig. 4A). Filipin, which only stains unesterified cholesterol, was used to check whether free cholesterol was increased in LAMP-2 deficient liver. Filipin staining of liver sections showed that LAMP-2 deficient liver accumulated unesterified cholesterol (Fig. 4A). The results indicate that, similar to LAMP-2 single deficient and LAMP double deficient MEFs, LAMP-2 deficiency leads to an accumulation of free cholesterol and increase in total cholesterol also in liver tissue. This finding suggests that LAMP-2 is more critical than LAMP-1 for the liver cholesterol homeostasis..


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)

Expression of LAMP-2influ-ences cholesterol levels. (A) LAMP-2 deficient liver accumulates cholesterol. Left panel: Total cholesterol was estimated from five wild-type, three LAMP-1–/–, and three LAMP-2−/− livers. The error bars represent standard deviation. The difference between the control and / LAMP-2−/− is statistically significant (P < 0.001). Middle and right panels: Filipin staining of liver sections from six-month-old wild-type / and LAMP-2−/− mice. (B) Gel filtration analysis of plasma lipoprotein / profiles of LAMP-2−/− mice and heterozygote littermate controls (please note that lamp-2 gene is located in the Y chromosome). Total cholesterol levels in the fractions are shown. Plasma samples from four mice were pooled for each group. Note that both female and male / LAMP-2−/− mice show elevated LDL cholesterol levels. (C, D) Overexpression of LAMP-2A retards the U18666A induced cholesterol accumulation. (C) HeLa cells were transfected with mouse LAMP-2A and grown for 29 hrs. The cells were then treated with increasing concentrations of U18666A for 15 hrs, and processed for filipin staining and immunolabelling with antimouse LAMP-2. Asterisks indicate the LAMP-2A overexpressing cells in the left panels. (D) The percentage of cells with late endosomal/lysosomal cholesterol accumulation was estimated. None, HeLa cells with no overexpression; mLA-2A, HeLa cells overexpressing mouse LAMP-2A. The results are the mean and standard deviation from one experiment with two parallel samples. The experiment was repeated with similar results.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822795&req=5

fig04: Expression of LAMP-2influ-ences cholesterol levels. (A) LAMP-2 deficient liver accumulates cholesterol. Left panel: Total cholesterol was estimated from five wild-type, three LAMP-1–/–, and three LAMP-2−/− livers. The error bars represent standard deviation. The difference between the control and / LAMP-2−/− is statistically significant (P < 0.001). Middle and right panels: Filipin staining of liver sections from six-month-old wild-type / and LAMP-2−/− mice. (B) Gel filtration analysis of plasma lipoprotein / profiles of LAMP-2−/− mice and heterozygote littermate controls (please note that lamp-2 gene is located in the Y chromosome). Total cholesterol levels in the fractions are shown. Plasma samples from four mice were pooled for each group. Note that both female and male / LAMP-2−/− mice show elevated LDL cholesterol levels. (C, D) Overexpression of LAMP-2A retards the U18666A induced cholesterol accumulation. (C) HeLa cells were transfected with mouse LAMP-2A and grown for 29 hrs. The cells were then treated with increasing concentrations of U18666A for 15 hrs, and processed for filipin staining and immunolabelling with antimouse LAMP-2. Asterisks indicate the LAMP-2A overexpressing cells in the left panels. (D) The percentage of cells with late endosomal/lysosomal cholesterol accumulation was estimated. None, HeLa cells with no overexpression; mLA-2A, HeLa cells overexpressing mouse LAMP-2A. The results are the mean and standard deviation from one experiment with two parallel samples. The experiment was repeated with similar results.
Mentions: We showed previously that, in LAMP−/− MEFs, the filipin staining (detecting free cholesterol) was increased and the level of total cholesterol was approximately twofold higher than in wild-type MEFs [15]. In LAMP-2 single deficient MEFs, the filipin staining intensity and cholesterol level were intermediate to wild-type and LAMP−/− MEFs. In order to test whether this phenotype would be observed in LAMP-2 deficient tissue, we investigated cholesterol levels in the liver of mice deficient in LAMP-1 or LAMP-2. Quantitative cholesterol assay showed that the level of total cholesterol was approximately twofold in the LAMP-2 deficient liver compared to the wild-type liver, whereas the level of LAMP-1 deficient liver was not different from the wild-type (Fig. 4A). Filipin, which only stains unesterified cholesterol, was used to check whether free cholesterol was increased in LAMP-2 deficient liver. Filipin staining of liver sections showed that LAMP-2 deficient liver accumulated unesterified cholesterol (Fig. 4A). The results indicate that, similar to LAMP-2 single deficient and LAMP double deficient MEFs, LAMP-2 deficiency leads to an accumulation of free cholesterol and increase in total cholesterol also in liver tissue. This finding suggests that LAMP-2 is more critical than LAMP-1 for the liver cholesterol homeostasis..

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