Genetics: Early Online, published on December 17, 2019 as 10.1534/genetics.119.302900 1 An ABCG transporter functions in Rab localization and lysosome-related organelle 2 biogenesis in Caenorhabditis elegans 3 4 5 6 Laura Voss, Olivia K. Foster, Logan Harper, Caitlin Morris, Sierra Lavoy, James N. 7 Brandt, Kimberly Peloza, Simran Handa, Amanda Maxfield, Marie Harp, Brian King, 8 Victoria Eichten, Fiona M. Rambo and Greg J. Hermann 9 10 Department of Biology, Lewis & Clark College, Portland, Oregon, United States of 11 America 1 Copyright 2019. 12 Running Title: 13 WHT-2 localization of GLO-1 14 15 Key Words: 16 lysosome-related organelle, Rab GTPase, ABC transporter, membrane trafficking, C. 17 elegans 18 19 Corresponding Author: 20 Greg Hermann 21 Lewis & Clark College 22 Department of Biology 23 0615 SW Palatine Hill Rd. 24 Portland, OR 97219 25 USA 26 Phone: 503-768-7568 27 E-mail: [email protected] 28 2 29 ABSTRACT 30 ABC transporters couple ATP hydrolysis to the transport of substrates across 31 cellular membranes. This protein superfamily has diverse activities resulting from 32 differences in their cargo and subcellular localization. Our work investigates the 33 role of the ABCG family member WHT-2 in the biogenesis of gut granules, a 34 Caenorhabditis elegans lysosome-related organelle. In addition to being required for 35 the accumulation of birefringent material within gut granules, WHT-2 is necessary 36 for the localization of gut granule proteins when trafficking pathways to this 37 organelle are partially disrupted. The role of WHT-2 in gut granule protein 38 targeting is likely linked to its function in Rab GTPase localization. We show that 39 WHT-2 promotes the gut granule association of the Rab32 family member GLO-1 40 and the endolysosomal RAB-7, identifying a novel function for an ABC transporter. 41 WHT-2 localizes to gut granules where it could play a direct role in controlling Rab 42 localization. Loss of CCZ-1 and GLO-3, which likely function as a guanine nucleotide 43 exchange factor (GEF) for GLO-1, lead to similar disruption of GLO-1 localization. 44 We show that CCZ-1, like GLO-3, is localized to gut granules. WHT-2 does not direct 45 the gut granule association of the GLO-1 GEF and our results point to WHT-2 46 functioning differently than GLO-3 and CCZ-1. Point mutations in WHT-2 that 47 inhibit its transport activity, but not its subcellular localization, lead to the loss of 48 GLO-1 from gut granules, while other WHT-2 activities are not completely 49 disrupted, suggesting that WHT-2 functions in organelle biogenesis through 50 transport-dependent and transport-independent activities. 51 3 52 INTRODUCTION 53 Cell type-specific elaboration of the conventional endocytic pathway leads to 54 the generation of lysosome-related organelles (LROs). Well-studied LROs include 55 mammalian melanosomes, mammalian platelet alpha and dense granules, 56 Drosophila retinal pigment granules, and Caenorhabditis elegans gut granules 57 (Bowman et al. 2019). LROs share contents, such as the V-ATPase and LAMPs, with 58 conventional lysosomes. However, they often lack degradative hydrolases and 59 instead harbor factors that endow each type of LRO with specialized functions. 60 Despite having diverse contents, morphologies, and activities, most LROs utilize the 61 same evolutionarily conserved machinery for their biogenesis. Some of these 62 biogenesis factors have functions that are dedicated to LRO formation while others 63 also act within the conventional endolysosomal system (Bultema and Di Pietro 64 2013; Marks et al. 2013; Bowman et al. 2019). Defects in the factors promoting LRO 65 formation underlie rare human diseases including Hermansky-Pudlak and Chediak- 66 Higashi syndromes (Huizing et al. 2008). 67 C. elegans gut granules are intestinal cell-restricted LROs that are generated 68 early during embryonic development and persist into adulthood (Laufer et al. 1980; 69 Clokey and Jacobson 1986; Bossinger and Schierenberg 1992; Hermann et al. 2005). 70 They contain autofluorescent derivatives of anthranilic acid (Coburn et al. 2013), as 71 well as rhabditin (Cobb 1914), an optically active birefringent material whose 72 molecular identity is currently unknown. Gut granules function in metal and 73 metabolite storage (Roh et al. 2012; Lee et al. 2015; Chun et al. 2017), anabolic 74 reactions (Panda et al. 2017), and life span regulation (Folick et al. 2015). Gut 75 granule formation requires the function of the Rab32 family member GLO-1, and the 4 76 AP-3 (Hermann et al. 2005), BLOC-1 (Hermann et al. 2012), and HOPS complexes 77 (Delahaye et al. 2014). A BLOC-3 related complex composed of CCZ-1 and GLO-3 78 likely acts as a guanine nucleotide exchange factor (GEF) activating GLO-1 and 79 promoting its gut granule association (Delahaye et al. 2014; Morris et al. 2018). In 80 addition to these factors, whose homologues function in the formation of 81 mammalian and Drosophila LROs (Bowman et al. 2019), we have identified a role for 82 three different ABC transporters in the biogenesis of gut granules (Currie et al. 83 2007; Schroeder et al. 2007). 84 ABC transporters represent a superfamily of conserved proteins that often 85 couple ATP hydrolysis to the movement of ions, lipids, sugars, and peptides across 86 cell membranes (Holland et al. 2003). ABC transporters localize to diverse cellular 87 membranes, including conventional lysosomes and LROs (Ridsdale et al. 2011; 88 Chapel et al. 2013), and there is evidence that they play a role in the formation of 89 both types of organelles. ABC transporters in yeast function in homotypic lysosome 90 fusion and exhibit direct physical interactions with the membrane trafficking 91 machinery, including subunits of the HOPS complex (Paumi et al. 2009; Sasser et al. 92 2012; Sasser et al. 2013; Snider et al. 2013). In Dictyostelium, ABC transporters 93 promote trafficking through the conventional endolysosomal pathway (Brazill et al. 94 2001). The biogenesis of lamellar bodies and melanosomes, LROs found within 95 human lung alveolar cells and melanocytes, respectively, requires the function of 96 ABC transporters (Cheong et al. 2007; Bergam et al. 2018). In C. elegans, the MRP-4, 97 PGP-2, and WHT-2 ABC transporters function in the formation of gut granules 98 (Currie et al. 2007; Schroeder et al. 2007). MRP-4 also impacts the size and function 99 of conventional lysosomes (Schaheen et al. 2006; Huynh et al. 2016). 5 100 WHT-2 is an ABCG subfamily member that is homologous to the Drosophila 101 White protein (Sheps et al. 2004). Similar to White, which transports pigment 102 precursors into retinal pigment granules (Sullivan and Sullivan 1975; O'hare et al. 103 1984), WHT-2 is required for the accumulation of birefringent material within gut 104 granules (Currie et al. 2007). However, genetic interactions between wht-2 and gut 105 granule biogenesis mutants suggest that WHT-2 has a broader role in the formation 106 of gut granules (Currie et al. 2007; Levitte et al. 2010). Here we show that WHT-2 107 has significant functions in gut granule biogenesis that extend beyond the 108 accumulation of birefringent material. Notably, our work indicates that WHT-2 109 functions in the localization of Rab GTPases, a novel activity for an ABC transporter. 110 6 111 MATERIALS AND METHODS 112 C. elegans strains were cultured on NGM media seeded with E. coli strain OP50 at 113 22oC (Stiernagle 1999). All mutant alleles were generated in N2, which was used as 114 the wild-type strain. The following mutations were used: apt-7(tm920), ccz- 115 1(ok2182), cup-5(zu223), glo-1(zu437), glo-1(syb1102[gfp::glo-1]), glo- 116 1(syb1103[gfp::glo-1]), glo-2(tm592), glo-3(gk582755), glo-3(kx38), glo-3(kx94), lyst- 117 1(gk295717), mrp-4(cd8), sand-1(or552), unc-119(ed3), vps-18(tm1125), wht- 118 1(tm688), wht-2(gk891224), wht-2(syb668[K74M]), wht-2(syb669[K74M]), wht- 119 2(syb745[K74R]), wht-2(syb794[K74R]), wht-2(ok2775), wht-3(ok927), wht- 120 4(ok1007), wht-5(ok806), wht-6(ok882), wht-7(ok812), wht-8(ok3112). Wormbase 121 (www.wormbase.org) hosts descriptions of each allele. The following transgenes 122 were used: amIs4[cdf-2p::cdf-2::gfp; unc-119(+)] (Davis et al. 2009), jcpEx2[ced- 123 1p::ccz-1::yfp; unc-119(+); myo-2::gfp] (Nieto et al. 2010), kxEx9[glo-1p::gfp::glo-1; 124 Rol-6D] (Hermann et al. 2005), kxEx41[glo-3p::glo-3::gfp; Rol-6D] (Rabbitts et al. 125 2008), kxEx152[asm-1p::asm-1::mCherry; Rol-6D] (Levitte et al. 2010), 126 kxEx172[WRM0622AF09; Rol-6 D] (this work), kxEx252[vha-6p::gfp::glo-1(D132A); 127 Rol-6D] (Delahaye et al. 2014), kxEx272[glo-1p::gfp::glo-1(D132A); Rol-6D] (Morris et 128 al. 2018), kxEx286[wht-2(+); Rol-6D] (this work), kxEx287[wht-2(+); Rol-6D] (this 129 work), kxEx289[wht-2(K74R); Rol-6D] (this work), kxEx296[wht-2(K74M); Rol-6D] 130 (this work), kxIs5[glo-1p::mans::mCherry; unc-119(+)] (this work); kxIs15[glo- 131 1p::gfp::glo-1; unc-119(+)] (this work), pwIs50[lmp-1p::lmp-1::gfp; unc-119(+)] 132 (Treusch et al. 2004), sIs10428[wht-2p::gfp; dpy-5(+)] (Mckay et al. 2003). 133 134 Genetic Manipulations 7 135 Standard genetic tests showed that wht-2(ok2775) and wht-2(gk891224) led to 136 recessive zygotic effect defects in birefringent gut granule formation. 137 Extrachromosomal (Ex) and integrated (Is) transgenes, present in otherwise wild- 138 type strains, were moved into mutant backgrounds by crossing males homozygous 139 or heterozygous for the mutation with hermaphrodites containing the transgenes.