Mucolipin-1 (MCOLN1) is a lysosomal calcium channel that belongs to the transient receptor potential (TRP) superfamily. Loss-of-function mutations in MCOLN1 result in mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by severe mental and psychomotor retardation. We recently performed a split-ubiquitin yeast two-hybrid screen with the goal to identify novel binding partners of MCOLN1 that allow us to better understand the cellular function of this protein. The screen identified two members of the lysosome-associated protein transmembrane (LAPTM) family as novel interaction partners of MCOLN1. The cellular function of LAPTMs is still poor characterized but some evidence suggests that they may participate in the transport of small molecules across the lysosomal membrane. The binding between MCOLN1 and LAPTM members (LAPTMs) was confirmed by co-immunoprecipitation and yeast two-hybrid assays. In addition, MCOLN1 and LAPTMs extensively colocalize at late endosomes and lysosomes. Overexpression of LAPTM4b caused enlargement of lysosomes and defective lysosomal degradation, indicating that LAPTMs are important for proper lysosomal function. Interestingly, lysosomal swelling induced by LAPTM4b was rescued by expression of MCOLN1, suggesting a functional connection between the two proteins. Finally, depletion of endogenous LAPTMs by siRNA induced accumulation of concentric multi-lamellar structures and electron-dense inclusions that closely resemble the structures found in MLIV cells. These data provide new insight into the molecular mechanisms of MCOLN1 function and suggest a potential role for LAPTMs in MLIV pathogenesis. To further understand the possible participation of LAPTMs in the development of MLIV, it will be of great importance to determine whether MCOLN1 regulate the transport activity of LAPTMs. It has been recently reported that over-expression of LAPTM4b contributes to chemotherapy resistance in breast cancer by preventing transport of the anthracycline doxorubicin to the nucleus. We found that LAPTM4b mediates sequestration of doxorubicin into lysosomes, thus causing drug resistance. Based on this observation, we have developed a simple cellular assay to monitor the activity of LAPTM4b. This assay consists on measuring LAPTM4b-dependent transport of doxorubicin into lysosomes by different methods including analyzing the cellular distribution of the drug (lysosomal versus nuclear staining), cell viability, and caspase-3 activation. Importantly, our results show that depletion of MCOLN1, but not MCOLN3, significantly inhibits the transport activity of LAPTM4b. Moreover, fibroblasts obtained from MLIV patients show increase sensitivity to treatment with doxorubicin. Our experiments will improve our understanding of the molecular bases of MLIV and may lead to the identification of new targets for therapeutic intervention.
|Vergarajauregui, Silvia; Martina, Jose A; Puertollano, Rosa (2011) LAPTMs regulate lysosomal function and interact with mucolipin 1: new clues for understanding mucolipidosis type IV. J Cell Sci 124:459-68|