Rab activation by ABC proteins during lysosome-related organelle biogenesis Lysosome-related organelles (LROs) are cell type-specific compartments that share characteristics with ubiquitous conventional lysosomes, yet have specialized functions. LROs are evolutionarily conserved derivatives of the endosomal system that include acidocalcisomes in protozoan parasites, gut granules in C. elegans, and mammalian melanosomes and platelet dense granules. Disrupting LRO biogenesis leads to Hermansky-Pudlak Syndrome (HPS), which is characterized by partial albinism and decreased blood clotting. LROs often coexist with conventional lysosomes due to the activity of trafficking pathways that divert LRO cargo away from conventional endolysosomes. These pathways have received relatively little attention as a result of only being deployed in specific cell types. Identifying and characterizing the activity of genes that function in LRO biogenesis is essential for diagnosing and developing therapies to treat HPS. The goal of this project is to gain a better understanding of the trafficking pathways leading to LROs by investigating the mechanisms by which LRO cell-restricted factors promote LRO biogenesis. Genetic screens have identified many C. elegans genes that function in gut granule biogenesis with mammalian counterparts that also mediate the formation of LROs. A subset of these, including GLO-1, GLO-3, and WHT-2, are only expressed in cells that generate LROs and they appear to be functionally connected. GLO-3 likely acts with CCZ-1 as a guanine nucleotide exchange factor (GEF) that activates the GLO-1 Rab. Rab function absolutely depends upon being recruited to the correct cellular membrane, which is in part mediated by GEF activity. Surprisingly, the ABC protein WHT-2 functions similarly to the GLO-1 GEF; WHT-2 is a gut granule protein that promotes LRO formation and the accumulation of GLO-1 on LROs. ABC proteins, which are conserved from bacteria to humans, couple ATP hydrolysis to the movement of diverse substrates across cell membranes. While ABC proteins have been implicated in endocytic trafficking, they have not previously been shown to function in Rab targeting. The discovery that WHT-2 acts in GLO-1 localization reveals a novel and important activity for ABC proteins in both LRO formation and regulating the specificity or stability of Rab intracellular targeting. The mechanisms by which WHT-2 functions in these processes is investigated through three specific aims: (1) to determine whether WHT-2 acts upstream of the GLO-1 GEF or downstream of activated GLO-1, possibly as an effector, to promote the gut granule association of GLO-1; (2) to determine whether the membrane transport activity or a novel function of WHT-2 is responsible for its roles in LRO biogenesis and the gut granule accumulation of GLO-1; (3) to test whether the phenotypic similarities between wht-7(-) and wht-2(-) mutants results from WHT-2 functioning as a heterodimer with WHT-7 in LRO formation and the accumulation of GLO-1 on gut granules. Given their conservation, it is likely that the functional interactions between ABC proteins and LRO Rabs are a general feature of LRO formation.
Statement of Relevance to Public Health Disrupting lysosome-related organelle (LRO) biogenesis leads to Hermansky-Pudlak Syndrome (HPS). The C. elegans genes being investigated are homologous to human genes that cause HPS. The proposed studies will provide a better understanding of their function and will be important in diagnosing and developing therapies to treat HPS patients.
