Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.KEYWORDS CUP-5; ESCRT; lysosome; mucolipidosis type IV; TRPML1 M UCOLIPIDOSIS type IV (MLIV) is a neurodegenerative lysosomal storage disorder characterized by corneal clouding, achlorhydria, and psychomotor defects (Bach 2001;Altarescu et al. 2002). In MLIV patients, large lipid-rich vacuoles are found in many tissues, while psychomotor defects are thought to be due to neuronal cell death. MLIV is caused by mutations in MCOLN1, which encodes the human protein mucolipin-1/TRPML1; this protein belongs to the transient receptor potential cation channel family and is a nonselective cation channel (Bargal et al. 2000;Bassi et al. 2000;Sun et al. 2000;Laplante et al. 2002;Raychowdhury et al. 2004;Dong et al. 2008).Caenorhabditis elegans protein CUP-5 is the ortholog of human TRPML1 (Fares and Greenwald 2001b;Hersh et al. 2002). The phenotypes resulting from mutations in cup-5(null) mimic those found in MLIV patients: defective lysosomal degradation and the appearance of large vacuoles in most tissues (Fares and Greenwald 2001b;Schaheen et al. 2006a). In addition, this lysosomal dysfunction in the absence of CUP-5 leads primarily to the death of developing intestinal cells that results in embryonic lethality (Schaheen et al. 2006a). It is not known why developing intestinal cells die in C. elegans or why neurons die in MLIV patients. In C. elegans cup-5(null) mutants, the embryonic lethality is not solely due to cells undergoing apoptosis from starvation; when ATP levels are restored or when apoptosis is blocked, embryonic lethality is only partially rescued (14% of embryos hatch and arrest at the L1 larval stage) (Hersh et al. 2002;Schaheen et al. 2006a).We have shown that C. elegans CUP-5 and mammalian TRPML1 likely function in lysoso...