Mucolipins are a family of ion channels that belongs to the superfamily of transient receptor potential (TRP) channels. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3), that exhibit a common six-membrane-spanning topology. MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). MLIV is a lysosomal storage disorder characterized by acute neurological and ophthalmologic defects. Symptoms appear during the first year of life and include mental retardation, delayed motor milestones, achlorhydria, and visual problems such us corneal clouding, retinal degeneration, sensitivity to light, and strabismus. The accumulation of enlarged vacuoles that contain different types of undigested lipids is a hallmark of MLIV cells and has lead to the suggestion that MCOLN1 might regulate membrane trafficking at the late endocytic pathway. More specifically, MCOLN1 has been suggested to participates in several Ca2+-dependent processes including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Consistent with the proposed role of MCOLN1 in lysosomal function, we have previously described that the protein localizes to late endosomes/lysosomes. Two di-leucine motifs cooperate to regulate delivery of MCOLN1 to lysosomes through interactions with the clathrin adaptors AP1, AP2, and AP3. The C-terminal tail of MCOLN1 undergoes post-translational modifications that regulate its activity and trafficking. Thus, for example, palmitoylation of three cysteine residues (Cys565, Cys566, and Cys567) increases the rate of MCOLN1 internalization from the plasma membrane, while PKA-mediated phosphorylation of Ser557 and Ser559 negatively regulates MCOLN1 channel activity in vivo. We have also found that the alterations of the late endosomal/lysosomal pathway observed in MCOLN1 deficient cells cause defective autophagy and lead to the accumulation of protein inclusions and ubiquitinated aggregates that might contribute to the neurodegeneration observed in MLIV patients. To better understand the mechanisms that regulate MCOLN1 function we have searched for proteins that interact with MCOLN1 in a Ca2+-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca2+, and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37-49. In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that over-expression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosomes markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. These data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca2+ sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway. MCOLN3 shows about 75% amino acid similarity with MCOLN1. Mutations in MCOLN3 are the cause of the varitint-waddler phenotype in mice, characterized by hearing loss, vestibular dysfunction (circling behavior, head-bobbing, waddling), and coat color dilution. Whole cell patch clamp experiments revealed that TRPML3 is an inwardly (from lumen into cytoplasm) rectifying monovalent cation channel that is permeable to Ca2+ and suppressed by low pH. We have addressed the function of MCOLN3 in epithelial cells. We found that MCOLN3 primarily localizes to early and late endosomes in ARPE-19 cells. This distribution at the less acidic portions of the endocytic pathway is consistent with the reported inactivation of the channel by low pH. Furthermore, we found that over-expression of MCOLN3 caused dramatic alterations in the endosomal pathway, including enlargement of Hrs-positive endosomes, delayed degradation of EGF and EGFR, and defective autophagosome maturation. Interestingly, similar defects have been described upon over-expression of proteins implicated in endosome biogenesis, such as ESCRT and Hrs. We have also found that endosomal pH is higher in cells over-expressing MCOLN3 and propose a model in which Ca2+ release from endosomes mediated by MCOLN3 might be important for efficient endosomal acidification. Therefore, MCOLN3 is a novel Ca2+ channel that plays a crucial role in the regulation of cargo trafficking along the endosomal pathway. Overall, our data reveal new evidence indicating that mucolipins distribute to specific locations along the endosomal pathway and play an important regulatory role in the sorting of lipids and proteins.
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