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First published online 6 June 2006
doi: 10.1242/jcs.02993

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Cholesterol depletion facilitates ubiquitylation of NPC1 and its association with SKD1/Vps4

¹ Department of Neurobiology, Tottori University Faculty of Medicine, Yonago 683-8503, Japan
² Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Kyoto 606-8501, Japan
³ Department of Cell Genetics, National Institute of Genetics, Mishima 411-8540, Japan
⁴ Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA
⁵ INSERM Unit 189, Lyon-Sud Medical School and Fondation Gillet-Merieux, Lyon-Sud Hospital, 69921 Oullins, France
⁶ Department of Child Neurology, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

View larger version (84K): [in a new window]   Fig. 1. Effects of cholesterol depletion on ubiquitylation of Flag-NPC1 expressed in COS cells. (A) Cellular cholesterol levels. Cells were cultured in cholesterol-rich medium (DMEM + 10% BCS) or cholesterol-depleted medium (DMEM + 10% LPDS supplemented with compactin and mevalonate) with or without LDL (20 µg/ml) up to 24 hours. Concentrations of total cholesterol in cell lysates were determined as described in the Materials and Methods. Each point represents the mean of duplicated determinations obtained in a single experiment. (B) Conjugation of myc/His6-ubiquitin (myc/His6-Ubi) to Flag-NPC1. Cells were transfected with expression constructs for myc/His6-ubiquitin together with Flag-NPC1 or an empty vector. 48 hours after transfection, they were further cultured in the indicated medium. 0.5% CHAPS extracts were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting (IB) with indicated antibodies. Molecular weights are given on the left (kDa). The arrowhead indicates the top of the separating gel and the asterisk indicates the heavy chain. (C) Co-purification of Flag-NPC1 with myc/His6-ubiquitin. After incubation in the indicated medium for 6 hours, cell extracts were subjected to affinity purification with metal resin followed by immunoblotting with indicated antibodies. (D) Conjugation of endogenous ubiquitin to Flag-NPC1. Cells were transfected with Flag-NPC1 or an empty vector, cultured in the indicated medium for 6 hours and anti-Flag IP products were analyzed by indicated antibodies. (E) Effects of exogenous cholesterol on ubiquitylation of Flag-NPC1. Cells were transfected with myc/His6-ubiquitin and Flag-NPC1, and cultured for 6 hours in cholesterol-rich or cholesterol-depleted medium supplemented with increasing concentrations of cholesterol. (F) Effects of U18666A. The transfected cells were cultured in the indicated medium for 12 hours in the presence or absence of U18666A. (G) Solubility of Flag-NPC1 to 1% Triton X-100. The transfected cells were cultured in the indicated medium for 6 hours. Total cell extracts (T), 1% Triton X-100-soluble (S) and Triton X-100-insoluble (I) fractions were prepared as described in the Materials and Methods. In E-G, anti-Flag immunoprecipitation products were subjected to immunoblotting with anti-Flag or anti-myc. All results shown are representative and were reproduced at least twice.

View larger version (99K): [in a new window]   Fig. 2. Ubiquitylation of mutant NPC proteins. COS cells were transfected with myc/His6-ubiquitin (myc/His6-Ubi) together with Flag-NPC1 constructs that encoded the wild-type (wt) or mutant proteins P691S (A) or LLNF (B); the arrowhead indicates the top of the separating gel. Cells were cultured in the indicated medium for 6 hours (left panels) or treated with or without MG132 for 6 hours (right panels). In both A and B, anti-Flag immunoprecipitation (IP) products were subjected to immunoblotting (IB) with anti-Flag or anti-myc. All results shown are representative and were reproduced at least twice.

View larger version (103K): [in a new window]   Fig. 3. Effects of MG132 and leupeptin on ubiquitylation of Flag-NPC1. COS cells were transfected with myc/His6-ubiquitin (myc/His6-Ubi) and Flag-NPC1 constructs, and were cultured in the indicated medium for 6 hours in the absence or presence of MG132 (upper panel) or leupeptin (lower panel); the arrowhead indicates the top of the separating gel. Anti-Flag immunoprecipitation (IP) products were subjected to immunoblotting (IB) with anti-Flag or anti-myc. All results shown are representative and were reproduced at least twice.

View larger version (25K): [in a new window]   Fig. 4. Interaction between SKD1(E235Q) and NPC1. (A) Schematic representation of expressed SKD1 proteins. Each tag is shown in gray. (B) Flag-NPC1 ubiquitylation. COS cells were transfected with the Flag-NPC1 construct together with an empty vector or His6-SKD1 constructs. 48 hours after transfection, anti-Flag immunoprecipitation (IP) products were subjected to immunoblotting (IB) with anti-Flag or anti-ubiquitin (P4D1). Molecular weights are given on the left (kDa); the arrowhead indicates the top of the separating gel. (C) Intracellular localization of GFP-SKD1 and Flag-NPC1. COS cells expressing Flag-NPC1 and GFP-SKD1 wild-type (wt; upper) or the E235Q mutant (EQ; lower) were fixed and stained with anti-Flag antibody. Bound antibody was visualized with Alexa Fluor 546-conjugated secondary antibody. Results shown are the representative images obtained with a confocal microscope. (D) Co-precipitation of Flag-NPC1 and GFP-SKD1 proteins. 0.5% CHAPS extracts were prepared from COS cells transfected with Flag-NPC1 together with GFP-SKD1wt or E235Q constructs. Anti-Flag immunoprecipitation products were analyzed by immunoblotting with the indicated antibodies. Cell extracts (IN: input) were loaded on lanes 1, 3, 5 and 7, and corresponding immunoprecipitation products were loaded on lanes 2, 4, 6 and 8. (E) Co-purification of endogenous NPC1 in CHO cells with His6-SKD1 proteins. Upper; intracellular localization of GFP-SKD1. Results shown are the representative images obtained with a confocal microscope. Lower; affinity purification. 0.5% CHAPS extracts were prepared from cells transfected with an empty vector, His6-SKD1wt or E235Q constructs. His6-tagged proteins were affinity purified with metal affinity resin and were analyzed by immunoblotting with the indicated antibodies. Cell extracts (IN; input) were loaded on lanes 1, 3 and 5, and corresponding affinity-purification products (AP) were loaded on lanes 2, 4 and 6. (F) In vitro interaction between GST-SKD1 and Flag-NPC1. GST-fused proteins, as shown in the schematic representations, were expressed in E. coli and immobilized on glutathione sepharose (left, CBB: Coomassie Blue staining). 0.5% CHAPS extracts (input) were prepared from COS cells expressing Flag-NPC1. Cell extracts were incubated with immobilized GST-fused proteins in the absence or presence of 0.5 mM ADP, ATP or ATPs. Bound proteins were eluted with glutathione and analyzed by anti-Flag immunoblotting (right). Molecular weights are given on the left (kDa). All results shown are representative and were reproduced at least twice.

View larger version (37K): [in a new window]   Fig. 5. Effects of cholesterol depletion on an interaction between Flag-NPC1 and wild-type SKD1. Transfected COS cells were cultured in cholesterol-rich or cholesterol-depleted medium with or without LDL for 16 hours. (A) Co-precipitation of His6-SKD1wt with Flag-NPC1. Cells were transfected with His6-SKD1wt together with Flag-NPC1 constructs. Anti-Flag immunoprecipitation (IP) products were analyzed by immunoblotting (IB) with the indicated antibodies. Molecular weights are given on the left (kDa). (B) Cell fractionation. Cells were transfected with Flag-NPC1 and His6-SKD1wt constructs. Membrane fractions (100,000 g pellet) of cell homogenates were fractionated on an Opti-prep gradient. The fractions, together with the membrane fractions applied (input), were analyzed by immunoblotting with the indicated antibodies. Shown are representative results that were reproduced three times. (C) Intracellular localization of Flag-NPC1 and GFP-SKD1wt. Cells expressing Flag-NPC1 and GFP-SKD1wt were stained with anti-Flag antibody, and bound antibody was visualized with Alexa Fluor 546-conjugated secondary antibody. Results shown are the representative images obtained with a confocal microscope. Shown in the insets are the enlarged images of the indicated areas.

View larger version (37K): [in a new window]   Fig. 6. Effects of cholesterol depletion on ubiquitylation of endogenous NPC1 and its interaction with SKD1 in human skin fibroblasts. Cells from a control subject or patients with NPC1 or NPC2 disease were cultured in cholesterol-rich or cholesterol-depleted medium for 16 hours. (A) Expression of NPC1 and SKD1. 0.5% CHAPS extracts were analyzed by immunoblotting (IB) with antibodies against the indicated proteins. (B) NPC1 ubiquitylation and its association with SKD1. 0.5% CHAPS extracts were subjected to anti-ubiquitin (P4D1) immunoprecipitation (IP) and the immunoprecipitated products were analyzed by immunoblotting with the indicated antibodies. Molecular weights are given on the left (kDa). (C) Cell fractionation. Membrane fractions (100,000 g pellet) of cell homogenates were fractionated on an Opti-prep gradient. All results shown are representative and were reproduced at least twice.