Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.
ABC transporters couple ATP hydrolysis to the transport of substrates across cellular membranes. This protein superfamily has diverse activities resulting from differences in their cargo and subcellular localization. Our work investigates the role of the ABCG family member WHT-2 in the biogenesis of gut granules, a Caenorhabditis elegans lysosome-related organelle. In addition to being required for the accumulation of birefringent material within gut granules, WHT-2 is necessary for the localization of gut granule proteins when trafficking pathways to this organelle are partially disrupted. The role of WHT-2 in gut granule protein targeting is likely linked to its function in Rab GTPase localization. We show that WHT-2 promotes the gut granule association of the Rab32 family member GLO-1 and the endolysosomal RAB-7, identifying a novel function for an ABC transporter. WHT-2 localizes to gut granules where it could play a direct role in controlling Rab localization. Loss of CCZ-1 and GLO-3, which likely function as a guanine nucleotide exchange factor (GEF) for GLO-1, lead to similar disruption of GLO-1 localization. We show that CCZ-1, like GLO-3, is localized to gut granules. WHT-2 does not direct the gut granule association of the GLO-1 GEF and our results point to WHT-2 functioning differently than GLO-3 and CCZ-1. Point mutations in WHT-2 that inhibit its transport activity, but not its subcellular localization, lead to the loss of GLO-1 from gut granules, while other WHT-2 activities are not completely disrupted, suggesting that WHT-2 functions in organelle biogenesis through transport-dependent and transport-independent activities.
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