Academic journal article Genetics

ESCRT-Dependent Cell Death in a Caenorhabditis Elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

Academic journal article Genetics

ESCRT-Dependent Cell Death in a Caenorhabditis Elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

Article excerpt

MUCOLIPIDOSIS type IV (MLIV) is a neurodegenerative lysosomalstoragedisordercharacterizedbycornealclouding, 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 lysosome formation, which involves the budding of nascent lysosomes from endosomes and scission to release the nascent lysosomes (Treusch et al. 2004; Miller et al. 2015). In contrast to this outward budding event, Endosomal Sorting Complex Required for Transport (ESCRT) proteins are required for the sequestration of integral membrane proteins in intraluminal vesicles through an inward budding and scission event in late endosomes/multivesicular bodies (Henne et al. 2011). This ESCRT-dependent targeting of integral membrane proteins includes an early ubiquitination of the cargo and a late deubiquitination required for completion of the scission reaction for internalization into an intraluminal vesicle. This de-ubiquitination is carried out by a complex of ESCRTassociated proteins that include Did2p (Saccharomyces cerevisiae)/CHMP1b (human), Bro1p (S. cerevisiae)/Alix and HD-PTP (human), and Doa4p (S. cerevisiae)/USP8 [UBPY] (human) (Bowers et al. 2004; Reid et al. 2005; Mahul-Mellier et al. 2006; Nickerson et al. 2006; Richter et al. 2007; Row et al. 2007; Ali et al. 2013). Sequestered cargo is transported to lysosomes for degradation. However, the mechanisms coordinating the sequestration of cargo inside late endosomes and their subsequent transport to lysosomes are unknown.

We have previously shown that mutations in mrp-4 suppress the cup-5(null) lysosomal defect and embryonic lethality (Schaheen et al. 2006b). MRP-4 is a member of the ATP-binding cassette (ABC) transporter superfamily found in prokaryotes and eukaryotes that use ATP energy for transport of various molecules across membranes (Bauer et al. 1999; Dean et al. 2001; Sheps et al. 2004). Here, we show that reducing levels of worm ESCRT-associated proteins almost completely suppress the lysosomal defect and embryonic lethality due to the loss of CUP-5. Indeed, we show that, in the absence of CUP-5, misregulated ESCRTassociated protein activity results in altered ubiquitination of MRP-4, which leads to embryonic lethality. Our results implicate ESCRT-associated protein defects in cell death and tissue degeneration in this C. elegans model of MLIV.

Materials and Methods

C. elegans strains and methods

Standard methods were used for genetic analysis (Brenner 1974). RNAi was done by the feeding method; control RNAi was done using bacteria expressing the double-stranded RNA generating vector L4440/pPD129. …

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