Abstract
Mucolipin-1 (ML1) is a member of the transient receptor potential ion channel superfamily that is thought to function in the biogenesis of lysosomes. Mutations in ML1 result in mucolipidosis type IV, a lysosomal storage disease characterized by the intracellular accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, and mucopolysaccharides. Little is known about how ML1 trafficking or activity is regulated. Here we have examined the processing and trafficking of ML1 in a variety of cell types. We find that a significant fraction of ML1 undergoes cell type-independent cleavage within the first extracellular loop of the protein during a late step in its biosynthetic delivery. To determine the trafficking route of ML1, we systematically examined the effect of ablating adaptor protein complexes on the localization of this protein. Whereas ML1 trafficking was not apparently affected in fibroblasts from mocha mice that lack functional adaptor protein complex (AP)-3, small interfering RNA-mediated knockdown revealed a requirement for AP-1 in Golgi export of ML1. Knockdown of functional AP-2 had no effect on ML1 localization. Interestingly, cleavage of ML1 was not compromised in AP-1-deficient cells, suggesting that proteolysis occurs in a prelysosomal compartment, possibly the trans-Golgi network. Our results suggest that posttranslational processing of ML1 is more complex than previously described and that this protein is delivered to lysosomes primarily via an AP-1-dependent route that does not involve passage via the cell surface.
Highlights
Mucolipidosis type IV (MLIV) pathophysiology has been linked to mutations in the transient receptor potential (TRP) channel family member mucolipin-1 (TRPML subfamily; referred to as ML1), where mutations result in a defect in membrane sorting along the late endocytic pathway (4 – 6)
It is thought that this large extracellular loop may be involved in channel activation, since both ML1 and polycystin-2 have relatively short amino- and carboxyl-terminal cytoplasmic tails that often serve as activation regions for other cation channels
Mutations in the cup-5 gene have been described to cause a defect in lysosome biogenesis, since CUP-5 is localized to both late endosome-lysosome fusion sites as well as to mature lysosomes
Summary
Delivery of ML1 to lysosomes occurs via a direct pathway dependent on adaptor protein complex-1 (AP-1) and does not involve passage via the cell surface. The ML1Myc doublet observed upon N-glycanase treatment appears to be due to incomplete cleavage of the N-glycans on this fragment, since longer incubation results in conversion of the more slowly migrating form to the more rapidly migrating form of the protein.
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