Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic - PubMed (original) (raw)

Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic

Paul R Pryor et al. Traffic. 2006 Oct.

Abstract

Mucolipin-1 is a membrane protein encoded by the gene MCOLN1, mutations in which result in the lysosomal storage disorder mucolipidosis type IV (MLIV). Efficient lysosomal targeting of mucolipin-1 requires di-leucine motifs in both the N-terminal and the C-terminal cytosolic tails. We have shown that aberrant lactosylceramide trafficking in MLIV cells may be rescued by wild-type mucolipin-1 expression but not by mucolipin-1 mistargeted to the plasma membrane or by lysosome-localized mucolipin-1 mutated in its predicted ion pore-selectivity region. Our data demonstrate that the correct localization of mucolipin-1 and the integrity of its ion pore are essential for its physiological function in the late endocytic pathway.

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Figures

Figure 1

Figure 1. Mucolipin-1 is a lysosomal membrane protein.

(A) Immunoblot of rat mucolipin-1 in rat liver fractions enriched in late endosomes (LE) and lysosomes (L). Proteins (10 μg total per track) were separated by electrophoresis on a 10%SDS polyacrylamide gel using a discontinuous buffer and, after immunoblotting were visualised by ECL. (B) NRK cells were fractionated and the homogenate separated on a 1-22% Ficoll gradient with a 45% Nycodenz cushion. 3 ml fractions were collected from the bottom of the gradient. The refractive index (RI) of the gradient fractions is shown. Each fraction was assayed for β-hexosaminidase activity and immunoblotted for MPR, lgp110 and mucolipin-1. (C) Confocal images (shown as maximum intensity Z projections) using indirect immunofluorescence of (a)GFP-mucolipin-1, (b)lgp120 and (c)extent of co-localisation in NRK cells stably expressing GFP-mucolipin-1. Bar, 10 μm. (D) Immunoelectron micrograph showing GFP-mucolipin-1 (15 nm gold) and lgp110 (10 nm gold) in NRK cells stably expressing GFP-mucolipin-1. Bar, 100 nm.

Figure 2

Figure 2. Two di-leucine signals are required for efficient targeting of mucolipin-1 to lysosomes.

cDNAs encoding human mucolipin-1, single di-leucine mutants L577/578A and L15/16A, and a double di-leucine mutant L15/16A + L577/578A, were sub-cloned into pEGFP-C3 and were transiently expressed for 24h in NRK cells. Confocal images show the extent of co-localisation between the various GFP-mucolipin-1 constructs and lgp120 as indicated by yellow in the panels in the right hand column. Bars, 10 μm.

Figure 3

Figure 3. Aberrant LacCer trafficking in MLIV cells is rescued by expression of lysosome-localised mucolipin-1.

(A) Fluorescence images of LacCer in cells heterozygous for a mutation in the mucolipin-1 gene (parental cells, WG0987), in untransfected MLIV cells (WG0909) and MLIV cells (WG0909) transiently transfected for 24h with either HcRed-tagged mucolipin-1 (wt) or a double di-leucine mutant (L15/16A + L577/578A). Endocytosed LacCer was observed after 3 h uptake in untransfected or transfected cells, the latter identified by the presence of HcRed. (B) Quantitation of LacCer puncta (excluding Golgi fluorescence) in the parents' cells heterozygous for mutations in the mucolipin-1 gene (WG0987 & WG0988), the MLIV patient's cells (WG0909) and MLIV (WG0909) cells transfected with HcRed-tagged wildtype (wt) or double di-leucine mutant (L15/16A + L577/578A) mucolipin-1. Quantitation is from analysis of ≥10 cells for each cell type from ≥ 3 separate experiments. **, P< 0.01 (WG0909 vs. WG0909 + mucolipin-1 (wt)). (C) MLIV, WG0909 cells were pulsed for 1 h with BODIPY-LacCer and chased for 2 h. Both the pulse and chase were in the presence of 1 mg/ml Texas Red dextran. The enlarged region shows co-localisation (yellow) between the LacCer (green) and the dextran (red). (D) WG0909 cells were pulsed with Oregon green 488–dextran (green) for 4 h then chased for 20 h. Cells were fixed and stained for lamp-1 (red). Co-localisation is shown by yellow. Bar = 10 _μ_m.

Figure 4

Figure 4. Cholesterol distribution and LacCer traffic kinetics

(A) MLIV, WG0909 and parental, WG0987 cells treated for 24 h with or without U18666A were stained for unesterified cholesterol (filipin) and lamp-1. Merged pictures show lamp-1 (green) and filipin (red). Bars = 10 _μ_m. (B) Parental and MLIV fibroblasts were pulsed for 1 h with LacCer and then chased for either 2 or 5 h (top and middle panels respectively). Cells treated for 24 h with U18666A were also pulsed for 1 h with LacCer and then chased for 2 h (bottom panels). Bars = 10 _μ_m.

Figure 5

Figure 5. Transfection of MLIV cells with mucolipin-1 constructs containing mutations in the pore region fail to rescue LacCer trafficking.

(A) Alignment of part of the pore region of human mucolipin-1 (Muc-1) with members of the TRP super-family and a voltage-gated Ca2+ channel, CACNLIA1 (for references see Supplementary Information). Three or more identical residues are shaded blue. A conserved phenylalanine (shaded green) includes residue F465 of mucolipin-1 (*) which is mutated in at least one case of MLIV (2). Residues which have been previously shown to affect pore channel activity are coloured purple. Residues which we predicted to affect pore channel activity and which we subsequently mutated (aspartates 471 and 472) are coloured red. Accession numbers for sequences (in order) Q9GZU1, Q13563, Q8NER1, Q9HBC0, Q9NQA5, Q9H1D0, P48995, Q13936. (B) NRK cells were transiently transfected for 24h with cDNA encoding different HcRed-tagged mutated mucolipin-1 constructs, F465L, single aspartate mutant D471K or double aspartate mutant D471/472K. Confocal images show the extent of co-localisation between the HcRed-tagged mucolipin mutants and lgp120 as indicated by yellow in the panels in the right hand column. Bars, 10 μm. (C) Fluorescence images of LacCer and quantitation of LacCer puncta in MLIV WG0909 cells transfected with the HcRed-tagged mutant mucolipin-1 constructs F465L, D471K and D471/472K (data from WG0909 and WG0909+mucolipin-1 (wt) cells from Fig 3B is included for comparison). Quantitation is from analysis of ≥10 cells for each cell type from ≥ 3 separate experiments. ns (not significant, P>0.05; data compared to WG0909). **, P< 0.01 (WG0909 vs. WG0909 + mucolipin-1 (wt)).

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