Function and regulation of the Caenorhabditis elegans Rab32 family member GLO-1 in lysosome-related organelle biogenesis

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.

Introduction pulled down both full length CCZ-1 and the CCZ-1(1-200) amino terminal region (Fig 1B). GLO-3(1-219) was able to pull down CCZ-1 , albeit not as strongly as full length GLO-3 ( Fig 1B). The GST moiety attached to GLO-3 did not significantly interact with either form of CCZ-1 (Fig 1B). Taken together, these results show that GLO-3 and CCZ-1 directly interact and that the amino-terminal domain of CCZ-1, which contains a longin domain [40], acts as a binding interface between the two proteins.

GLO-1 and GLO-3 promote protein localization to gut granules and not lysosomes
We analyzed whether the effects of mutations in glo-1 and glo-3 on gut granule and endolysosome biogenesis resembled the loss of ccz-1 function. The glo-1(zu437) allele used in our studies completely lacks GLO-1 activity [1]. We have isolated a glo-3 allelic series spanning four phenotypic classes (I-IV) [44], ranging from the least severe, a newly identified class IV glo-3 (gk582755) missense allele GLO-3(N279K) that causes a moderate reduction in gut granule numbers, to the strongest class I alleles, represented by glo-3(kx94), which lack birefringent gut granules in embryos but retain a few autofluorescent gut granules in adults (Tables 1 and 2). Nearly all of the glo-3 alleles are premature stop codons and their location in glo-3 does not correlate with their phenotypic severity [44]. We note that the two class I alleles cause amber stop codons, while the weaker class II and class III alleles cause ochre or opal stop codons [44]. In C. elegans, opal and ochre stop codons can occasionally be read through during translation, while amber stop codons cannot [45], suggesting that the class II and III alleles produce differing amounts of full length GLO-3. CRISPR-Cas9 was used to delete the entire glo-3 coding sequence and the resulting glo-3(syb272) allele was phenotypically indistinguishable from class I mutants (S1 Fig and Tables 1 and 2), strongly suggesting that the glo-3(kx94) allele used in this work represents a null allele.
In prior work, we showed that disrupting the function of glo-1 or glo-3 leads to significant reductions in autofluorescent and birefringent gut granules (Tables 1 and 2) [1,19]. To determine if this is associated with defects in protein localization in glo-1(-) and glo-3(-) mutants, we analyzed the steady state distribution of the gut granule transmembrane proteins CDF-2, which functions as a Zn transporter [46], the ABC transporter PGP-2 [47], and the Lamp1 homologue LMP-1 [48]. CDF-2 and PGP-2 are restricted to gut granules, while LMP-1 is associated with both gut granules and conventional lysosomes [13]. To minimize indirect effects, we carried out our analyses in embryonic intestinal cells soon after gut granules are first generated [3]. In glo-1(zu437) and glo-3(kx94) mutants, the distribution of CDF-2::GFP and LMP-1 was dramatically altered (Fig 2B and 2C). Strikingly, both were mislocalized to the intestinal cell membrane (Fig 2B and 2C). While the extensive colocalization of CDF-2::GFP and LMP-1 was not disrupted in these mutants, they were not located on organelles that resembled gut granules (Fig 2B, 2C and 2G). Instead, the morphology and position of the compartments containing these markers were similar to conventional lysosomes. To examine whether CDF-2:: GFP was being mislocalized to lysosomes, we analyzed the distribution of CDF-2::GFP relative to an mCherry tagged form of GBA-3, a glucosylceramidase localized to degradative lysosomes that when disrupted in humans causes Gaucher disease [13,49,50]. We found that CDF-2:: GFP was mislocalized to conventional lysosomes in glo-1(-) and glo-3(-) mutants (Fig 2D, 2F and 2I), explaining why the colocalization of CDF-2::GFP and LMP-1 was not altered in these mutants. PGP-2 was lacking in both glo-1(-) and glo-3(-) mutants (Fig 2B, 2C and 2H), possibly due to its lysosomal mistargeting and degradation. These results indicate that GLO-1 and GLO-3 both play essential roles in the routing of proteins to gut granules, similar to what we have previously seen for CCZ-1 [19]. All strains were grown at 22˚C. Three-fold and later stage embryos were analyzed using polarization microscopy and scored for the presence and number of birefringent granules in the intestine. The expression of gfp::glo-1 was assayed with fluorescence microscopy. https://doi.org/10.1371/journal.pgen.1007772.t001 Function and regulation of GLO-1 We next addressed whether GLO-1 and GLO-3 function in the formation of lysosomes. Gut granules and conventional lysosomes are distinct and co-exist in embryonic intestinal cells [13]. Some factors that mediate gut granule biogenesis, including CCZ-1, also function in lysosome biogenesis and disrupting their activity causes a significant increase in the number of All strains were grown 22˚C. Adults were analyzed using fluorescence microscopy with a rhodamine filter to score the number of autofluorescent organelles within the intestine located posterior to the vulva and a FITC filter set to assess the expression of GFP::GLO-1. https://doi.org/10.1371/journal.pgen.1007772.t002 Function and regulation of GLO-1
We investigated whether sand-1(-) mutants disrupt the colocalization of CDF-2::GFP and LMP-1 similarly to ccz-1(-), due to the well-established role of the C. elegans CCZ-1/SAND-1 (MON1) complex in the biogenesis of late endosomes and trafficking to conventional lysosomes [42,56,57]. In sand-1(-) single mutants, the colocalization of CDF-2::GFP with LMP-1 was not obviously different than wild type (Fig 4B and 4L). However, gut granule biogenesis is with confocal microscopy. GBA-3::mCherry did not localize to CDF-2::GFP marked compartments in wild type (white arrowheads in insets), however it often did (white arrows within insets) in glo-1 and glo-3 mutants. (G-H) For each genotype, 20 randomly selected CDF-2::GFP containing intestinal compartments in 5 different embryos were scored for the presence of LMP-1 or PGP-2 signals. (I) For each genotype, 25 randomly selected CDF-2::GFP containing intestinal compartments in 5 different 1.5-fold stage embryos were scored for the presence of GBA-3::mCherry. In all images, a single optical section is shown and black arrowheads flank the intestine. Embryos are 50μm in length. In all graphs, the mean is plotted and error bars represent the 95% confidence limit. A one way ANOVA comparing each mutant to wild type was used to calculate p-values ( �� represents p � 0.001).

GLO-1 functions as a Rab GTPase to promote gut granule biogenesis
GLO-1 is a Rab32 family member and the conservation of G-motifs suggests that it functions as a GTPase [21,22]. To determine if GTP binding is important for the activity of GLO-1 in vivo we expressed GLO-1 point mutants in glo-1(zu437) animals. Whereas GFP tagged GLO-1 (+) restored gut granules in glo-1(zu437) (Fig 5A-5C), expression of GFP::GLO-1(T25N), which is predicted to disrupt GTP but not GDP binding [59], did not rescue the loss of autofluorescent, birefringent, and PGP-2 marked gut granules (Fig 5E and Tables 1 and 2). Expression of GFP::GLO-1(Q71L), which is predicted to lack GTP hydrolysis and maintain an active GTP-bound conformation [59], was able to functionally replace GLO-1(+) (Fig 5D and Tables  1 and 2). Neither GLO-1 point mutant dominantly disrupted gut granule biogenesis (Tables 1  and 2). The different mutant effects suggest that the GTP bound form of GLO-1 is active in promoting gut granule biogenesis.

GLO-3 and CCZ-1 act upstream of GLO-1
Mutations in the Rab G4 motif can weaken its affinity for guanine nucleotides leading to increased rates of nucleotide exchange that can bypass the requirement of a Rab for its corresponding GEF [41,60,61]. GLO-1(D132A) and GLO-1(I133F) G4 motif mutations restore autofluorescent compartments in ccz-1(-) and glo-3(-) mutant adults [19], suggesting that spontaneous nucleotide exchange bypasses the requirement for CCZ-1 or GLO-3 in gut granule biogenesis. The vha-6 promoter used in our prior study initiates expression late in embryogenesis [62,63]. To assess the effects of the GLO-1 G4 mutants at a stage when we can rigorously assess the biogenesis of gut granules using multiple organelle markers, we placed the point mutants under control of the glo-1 promoter, which leads to earlier intestinal expression. When introduced into glo-1(zu437) mutants, both GLO-1 G4 mutants restored birefringent compartments in embryonic intestinal cells and autofluorescent intestinal organelles in adults (Fig 6A-6D and Tables 1 and 2). We focused our analysis on GLO-1(D132A) as it had the strongest rescuing activity (Tables 1 and 2). GLO-1(D132A) restored birefringent and autofluorescent granules in glo-3(-) and ccz-1(-) mutants, but did not suppress the loss of these organelles in AP-3, BLOC-1, or HOPS mutants (Fig 6F and 6J and Tables 1 and 2). Expression of GLO-1(+) only restored birefringent and autofluorescent organelles in glo-1(-) mutants ( Fig  6C, 6E and 6I and Tables 1 and 2). Therefore, GLO-3 and CCZ-1 likely function upstream of GLO-1 in the formation of gut granules.
We investigated whether GLO-1(D132A) restored gut granule protein localization in glo-1 (-), glo-3(-), and ccz-1(-) mutants. For these experiments, we quantified the colocalization of proteins using SQUASSH image analysis software that deconvolves, segments, and calculates the overlapping area between two fluorescence signals in three dimensions [64]. This software enabled a comprehensive, high throughput, and quantitative approach for identifying and measuring the area of overlap between two different organelle markers within the entire embryonic intestine. The output C size (marker 1 marker2 /marker 1) describes the proportion of marker 1's area that contains marker 2.

GLO-3 and CCZ-1 localize GLO-1
Inactive Rabs are GDP-bound and can be extracted from organelle membranes into the cytosol by Rab GDI, while activated, GTP-bound Rabs are resistant to extraction and membrane localized [29,31]. The restoration of gut granules in glo-3(-) and ccz-1(-) mutants by GLO-1 (D132A) is consistent with GLO-3 and CCZ-1 functioning upstream of GLO-1 activation. We therefore examined whether GFP::GLO-1 was cytoplasmic and diffusely localized or enriched on organelles. For these experiments, we examined living embryos by imaging GFP::GLO-1 and performed line intensity scans to compare the organelle and cytoplasmic signals. In wild type, GFP::GLO-1 was localized to gut granules and relatively little signal was found in the cytoplasm (Fig 8A and 8I). When GFP::GLO-1 was identically imaged, both glo-3(-) and ccz-1 were stained with antibodies to PGP-2 and LMP-1. Embryos were imaged with confocal microscopy, single optical sections are shown, and white arrows in the insets label organelles containing the gut granule protein PGP-2. Black arrowheads flank the intestine. (E-F) SQUASSH software was used to calculate C size (PGP-2 LMP-1 or GFP::GLO-1 /PGP-2), which represents the proportion of total PGP-2 area in each embryo that also contained LMP-1 (E) or GLO-1::GFP (F) signals. Each data point represents the C size of an individual embryo and at least 5 embryos of each genotype were scored. The mean level of colocalization per embryo is plotted, bars represent the 95% confidence intervals, and �� indicates p�0.005, � indicates p�0.05, and ns indicates p>0.05, by one way ANOVA followed by a Tukey-Kramer test.
The class III glo-3(kx38) allele, which generates gut granules marked by PGP-2 (see next section), was used to address whether GLO-1 was localized to gut granules when glo-3 function was partially, but not completely, disrupted. In fixed wild-type embryos, GFP::GLO-1 was associated with gut granules, whereas it was lacking from PGP-2 marked gut granules in the glo-3(kx38) mutant (Fig 7F and S2). To test whether the ectopically expressed and epitope tagged GFP::GLO-1 behaves similar to endogenous GLO-1, we stained wild-type and glo-3 (kx38) embryos with anti-GLO-1 antibodies. In wild type, GLO-1 localized to gut granules marked by CDF-2::GFP (Fig 8J and 8L). In contrast, GLO-1 was lacking from gut granules in glo-3(kx38) mutants (Fig 8K and 8L). Taken together these results show that GLO-3 and CCZ-1 promote the association of GLO-1 with gut granules.

Gut granules in glo-3(kx38)
All of the glo-1 alleles we have isolated and the glo-3 class I null alleles, including glo-3(kx94), completely disrupt embryonic gut granule biogenesis (Figs 2 and 6 and Table 1) [1,44], making it difficult to determine how GLO-1 and GLO-3 function in the pathways generating gut granules. We have isolated a large number of glo-3(-) mutants with varying levels of glo-3(+) activity and a range of phenotypic effects (Tables 1 and 2) [44]. This allelic series can reveal phenotypes that result from partial glo-3(+) activity and point to specific functions for GLO-3 and the GLO-1 Rab it regulates in gut granule biogenesis.
While gut granules are present in glo-3(kx38) embryos, our work shows that both GFP:: GLO-1 and endogenous GLO-1 are lost from these compartments (Figs 7F and 8K and S2). Therefore an analysis of this mutant can reveal effects on LRO biogenesis when glo-3(+) activity is reduced and GLO-1 is lacking from gut granules. The most obvious effect of glo-3(kx38) is on gut granule number and size; compared to wild type, the number of gut granules marked by PGP-2 was reduced by more than 80% and the average gut granule diameter was 55% larger (Fig 9F and 9G). To determine if glo-3(kx38) disrupts protein trafficking, we analyzed the localization of gut granule markers in glo-3(kx38) mutants. LMP-1 is localized to both gut granules and conventional lysosomes [13], and in glo-3(kx38) mutant embryos LMP-1 was mislocalized to the plasma membrane and lacking or only weakly associated with gut granules (Fig 9C-9E). CDF-2::GFP remained associated with gut granules in glo-3(kx38) mutants ( Fig  9B and 9E). However, CDF-2::GFP was mislocalized to the plasma membrane and what are likely conventional lysosomes based upon their morphology, location, and enlargement in cup-5(-) mutants (Figs 9B and S3). These analyses show that the localization of CDF-2::GFP and LMP-1 are sensitive to reduction in glo-3 activity, while PGP-2 appears to be unaffected. Notably, the presence of gut granules in glo-3(kx38) mutants indicates that the enrichment of GLO-1 on gut granules is not necessary for their biogenesis.
We addressed whether RAB-7 has a role in the formation of gut granules in glo-3(kx38) mutants. Suggesting that it does not, we found that the formation of birefringent gut granules was not disrupted in glo-3(kx38); rab-7(ok 511) double mutants (Table 3). sand-1(-) mutations the entire intestine of 1.5-fold stage embryos stained with anti-PGP-2 antibodies were acquired. SQUASSH software was used to quantify the number of PGP-2 compartments in 5 embryos of each genotype. (E-G) In each graph the mean is plotted, bars represent the 95% confidence intervals, � indicates p�0.05, and �� indicates p�0.001 by one way ANOVA comparing glo-3(kx38) to wild type. https://doi.org/10.1371/journal.pgen.1007772.g009 Function and regulation of GLO-1
To further investigate the functional relationships between GLO-1 and RAB-7 we ectopically expressed GFP::GLO-1(+) and GFP::GLO-1(D132A) in glo-3(kx38) mutants and found that both led to a significant decrease in the association of RAB-7 with gut granules (Fig 10I-10M). These results point to a role for GLO-1 in preventing the association of RAB-7 with gut granules and suggest that the association of RAB-7 with gut granules in glo-3(kx38) mutants could result from the loss of GLO-1 from these organelles. One mechanism by which GLO-1/ GLO-3 could restrict RAB-7 from gut granules would be through the recruitment and/or activation of the RAB-7 GTPase activating protein (GAP). Currently, the RAB-7 GAP is not known in C. elegans, however the RAB-5 GAP TBC-2 has RAB-7 GAP activity in vitro, and genetic studies are consistent with it functioning as a RAB-7 GAP [69][70][71]. However, tbc-2(-) mutants did not lead to the mislocalization of RAB-7 to gut granules or obvious defects in gut granule protein trafficking (S6 Fig).

Discussion
GLO-1 and related Rab32/38 proteins were initially identified due to their role in the biogenesis of LROs in mammals [24,27], Drosophila melanogaster [25], and C. elegans [1]. More recently Rab32 family members have been implicated in autophagy [72,73], phagocytosis of bacterial pathogens [74][75][76][77], and endocytosis and proteolytic degradation [78]. In C. elegans, glo-1(-) early embryos are defective in the autophagic degradation of paternal mitochondria [79][80][81]. In the nervous system, glo-1(-) adults show decreased numbers of RAB-7 labeled compartments [82], altered necrosis [83], and defects in synapse formation and neuronal morphology [84]. All of these more recently identified roles for Rab32 family members, including GLO-1, could result from functions in the conventional endolysosomal pathway. In fact, many of the factors originally characterized as having a role in LRO biogenesis are now known to support conventional endolysosomal trafficking [1,19,[85][86][87][88][89]. Notably, we did not detect a significant role for GLO-1 in the transport of cargo through conventional endolysosomes and instead show that GLO-1 functions to direct protein cargo away from this pathway toward gut granules (Figs 2 and 3). Our data support an LRO restricted role for GLO-1 in intestinal cells and we suggest that processes mediated by gut granules impact developmental and physiological processes outside the intestine or that other C. elegans cell stained with anti-GLO-1 antibodies and imaged with confocal microscopy. GLO-1 was similarly associated with gut granules in wild type and rab-7(-) mutants (white arrows in insets). (H) SQUASSH software was used to calculate the area of CDF-2::GFP organelles that contained GLO-1 in 5 embryos of each genotype. The mean is plotted, bars represent the 95% confidence intervals, ns indicates p>0.05 by one way ANOVA. (I-L) Signals from antibodies to PGP-2 and RAB-7 were acquired in 1.5-fold stage embryos using confocal microscopy. The expression of GFP::GLO-1(+) or GFP::GLO-1(D132A) led to the diminished localization of RAB-7 on PGP-2 compartments in glo-3(kx38) mutants. (M) SQUASSH software was used to calculate the area of PGP-2 organelles that contained RAB-7 in 5-7 embryos of each genotype. The mean is plotted, bars represent the 95% confidence intervals, � indicates p�0.05 and �� indicates p�0.001 by one way ANOVA. In all images single optical sections are shown and black arrowheads flank the intestine. https://doi.org/10.1371/journal.pgen.1007772.g010 Function and regulation of GLO-1 types possess LROs, with different functions than gut granules, whose formation requires GLO-1.
Similar to other Rabs [29,31], we show that the GTP bound form of GLO-1 is active in gut granule formation (Fig 5). Following GTP hydrolysis, most Rabs will remain in the inactive form due to their low intrinsic rate of exchange of GDP for GTP [90]. In mammals, Rab32/38 guanine nucleotide exchange is catalyzed by BLOC-3, a heterodimeric complex composed of HPS1 and HPS4 [33]. BLOC-3 subunits show sequence and functional homology with CCZ-1/ SAND-1(MON1), which function as a heterodimeric GEF for RAB-7 [33,38,39,41,42,91,92]. In addition to interacting with SAND-1(MON1), we find that CCZ-1 can directly bind to GLO-3 (Fig 1). We have previously shown that CCZ-1, but not SAND-1 or RAB-7, is required for gut granule biogenesis, and that a point mutation in GLO-1 predicted to increase the rate of spontaneous guanine nucleotide exchange restores of autofluorescent organelles in ccz-1(-) and glo-3(-) mutant adults [19]. Here we definitively show that the function of CCZ-1 and GLO-3, but not other gut granule biogenesis factors, is bypassed by the GLO-1 fast-exchange mutant (Figs 6 and 7 and Tables 1 and 2). Furthermore, GFP::GLO-1 is diffusely localized in ccz-1(-) and glo-3(-) mutants and GLO-3 functions in the recruitment to and/or stabilization of GLO-1 on gut granules (Fig 8). Loss of GLO-1 GEF activity should result in the accumulation of GLO-1 in the GDP bound form, which would be extracted from organelle membranes into the cytosol by Rab GDI [29,31]. Together, these results strongly suggest a CCZ-1 and GLO-3 function as a GEF that activates and localizes GLO-1.
While the GLO-1(D132A) fast-exchange mutant promoted the proper localization of some gut granule proteins in glo-3(-) and ccz-1(-) mutants, the localization of GLO-1(D132A) was reduced and LMP-1 was noticeably absent from these organelles (Fig 7). Rab GEFs are known to play important roles localizing their Rab substrates, and it is currently unknown whether this is purely through catalyzing nucleotide exchange or through other functions such as physical interactions that recruit the Rab [29,31]. Our observations suggest the latter possibility for GLO-3 and CCZ-1. It is also possible that the higher level of GLO-1(D132A) gut granule association when GLO-3 and CCZ-1 are present, could result from these factors promoting the GTP, membrane localized form of the fast exchange mutant. LMP-1 trafficking to gut granules requires the function of the AP-3 adaptor complex, while other gut granule proteins can localize to gut granules independently of AP-3 [13]. The defects in LMP-1 localization could result from GLO-3 and CCZ-1 functioning in the AP-3 pathway independently of regulating GLO-1. The Rab GEFs, Rabin8, VARP, and possibly the TRAPP complexes, have GEF-independent roles in membrane dynamics [93][94][95]. Additionally, the activation cycle of GLO-1(D132A) might not fully restore wild-type GLO-1 activity, disrupting the delivery of LMP-1 to gut granules. In support of this possibility, biochemical and genetic analysis of analogous fast-exchange mutations in RAB-7 show that they cause decreased RAB-7 function [61,96].
Rab GEFs are currently thought to be the major factors determining the subcellular localization of Rabs [33,44,[109][110][111][112]. GLO-3 associates with gut granules [44], putting it in the correct position to direct GLO-1 localization. However, if GLO-3 and CCZ-1 function as a GLO-1 GEF, how does GLO-1(D132A) localize to gut granules in the absence of these proteins? Analogous fast exchange Rab7 and RAB-2 mutants are properly localized when the activity of their respective GEFs is lacking [113,114], suggesting that GEFs are not essential for the localization of Rabs that have an increased rate of nucleotide exchange. We know little about the identity and function of factors other than GEFs that impact the recruitment and/or stabilization of most Rabs, but they have been suggested to include Rab-GDI displacement factors or Rab effectors [115][116][117][118].
It is likely that each Rab utilizes a distinct set of interacting factors and mechanisms to ensure its correct spatiotemporal distribution [29,31]. Rab GEFs are typically not membrane anchored, a key characteristic of a membrane targeting receptor. Possibly there are integral membrane proteins that function as Rab receptors or modify the organelle membrane to promote Rab localization. The identification and characterization of these factors will be critical for understanding how organelles acquire their functional identity.

Genetic manipulations
Integrated (Is) and extrachromosomal (Ex) transgenes, present in otherwise wild-type strains, were moved into mutant backgrounds by crossing hermaphrodites containing the transgenes with males homozygous or heterozygous for the mutation. The presence of the mutation in the resulting strain was confirmed by the presence of the mutant phenotype, or in cases where this was modified by the transgene, by PCR and/or DNA sequencing. To generate double mutants containing glo-3(kx38), transheterozygous individuals were allowed to self fertilize and progeny that were homozygous for glo-3(kx38), as evidenced by the number of birefringent gut granules, and heterozygous for the other mutation, were isolated. The homozygous double mutants that exhibited the other mutant phenotype were then isolated from these strains. In cases where one Glo phenotype masked another, we confirmed the presence of the masked mutation using PCR/DNA sequencing. In all cases, single and double mutants were homozygous for each mutation except strains containing rab-7(ok511), cup-5(zu223), and some strains containing ccz-1(ok2182), which were kept heterozygous due to the recessive maternal effect lethality or severe growth defect caused by these mutations [54,57,86]. In cases where strains heterozygous for these mutations were used, we identified homozygous mutant adults by the presence of large DIC refractile granules within embryos in their uterus or a linked recessive marker. Mutant embryos produced by homozygous rab-7(-), cup-5(-), and ccz-1(-) parents display these morphologically distinct structures [54,57,86]. unc-36(e251) was linked to cup-5(-) and we found that it did not alter gut granule biogenesis in any of our assays. glo-3(gk582755) was identified in an ongoing screen of Million Mutation strains for defects in gut granule number and/or morphology. The Glo phenotype of strain VC40338 mapped to the X chromosome and did not complement the Glo phenotypes of glo-3(zu446). The glo-3(gk582755) mutation causes a GLO-3(N279K) substitution and was backcrossed 3 times to N2 before being characterized. CRISPR-Cas9 gene editing was carried out by SunyBiotech (Fuzhough City, Fujian, China) to generate glo-3(syb272), which precisely removes the entire glo-3 coding sequence. Sanger sequencing verifying the presence of the deletion was carried out by Genewiz (South Plainfield, NJ, USA). The resulting sequence TTCgAGGTAAACTCGTTCAAA-ATAATT-TATATTTACAAGTAT flanked the deletion (marked by-). The g denotes a mutation created to destroy the PAM site. syb272 was backcrossed 4 times to N2 before being characterized. To knock down the expression of rab-7 we used RNAi feeding protocol 1 described in [122] and clones from the Ahringer RNAi library (Source Bioscience, Nottingham, UK). The effects of rab-7 RNAi were not seen in embryos treated with F33E2.4(RNAi), which targets a gene not required for gut granule biogenesis. In RNAi experiments, inhibition of rab-7 activity was confirmed by the presence of DIC refractile granules.

Microscopy
Widefield polarization and fluorescence microscopy was carried out with a Zeiss AxioImager. M2 and images were captured with an AxioCam MRm digital camera controlled by AxioVision 4.8 software (Zeiss, Thornwood, NY). Confocal fluorescence microscopy was carried out with a Zeiss LSM710 laser scanning confocal microscope. Embryos were imaged with 100X or 63X Plan-Apochromat 1.4 NA objectives and adults were imaged with a 40X Plan-Apochromat 1.3 NA objective.
Adults were mounted on 3% agarose pads and immobilized with 10mM levamisole (Sigma Aldrich, St. Louis, MO). Autofluorescent gut granules were imaged with a Zeiss 38 filter (GFP, excitation, BP 470/40; emission, BP 525/50), a Zeiss 45 filter (mCherry, excitation, BP 560/40; emission, BP 630/75), or a 488 laser line. Z-stacks of the intestine were captured and maximum intensity projections of ½ or the entire depth of the intestine are shown. In adults expressing GFP tagged proteins, the Zeiss 45 filter was used to visualize gut granules.
Living embryos were mounted in H 2 O on 3% agarose pads. Body movements in embryos do not begin until after the 1.5-fold stage. To acquire images of late stage embryos, excess respiring OP50 bacteria was added to induce hypoxia and immobilization. Birefringent material was visualized with polarization optics. Maximum intensity projections of Z-stacks capturing all of the birefringent material within the intestine are shown. GFP and mCherry markers were imaged by confocal microscopy in living 1.5-fold stage embryos using the 488 and 561 laser lines. GFP, mCherry, and autofluorescence in living embryos were imaged using widefield microscopy with Zeiss, 38, Zeiss 45 and Zeiss 49 (DAPI, excitation, G 365; emission, 445/ 50) fluorescence filters, respectively.
To characterize the pattern of GFP::GLO-1 in living embryos, GFP::GLO-1 signals in each strain were captured using widefield fluorescence microscopy using identical exposure settings. Z-stacks through the top half of the intestine were captured. The Fiji software plot profile tool centered on randomly selected puncta was used to generate intensity profile histograms [123]. The intensity value for each punctum was calculated by subtracting the average of the 10 lowest intensity values from the peak value in each 60 pixel intensity histogram. Widefield fluorescence Z-stacks of autofluorescent gut granules in pretzel stage embryos were imaged with a Zeiss 49 filter. The diameter of these organelles was determined using Zeiss AxioVision software.
Embryos were fixed in -20˚C MeOH following a freeze-crack as described [124]. The intrinsic fluorescence of GFP was used to visualize the distribution of GFP tagged proteins after fixation. Antibodies to GLO-1 [1], LMP-1 [125], PGP-2 [47], RAB-5 [126], and RAB-7 [127] were used. Z-stacks through the intestine of fixed LMP-1::GFP expressing embryos were imaged using widefield microcopy with a Zeiss 38 filter. Using the plasma membrane localization of LMP-1::GFP to identify individual cells, we manually quantified the number of lysosomes located within the 4 cells that make up Int 2 and 3. To simultaneously image the localization of CDF-2::GFP, PGP-2, and LMP-1 in embryos, we used secondary antibodies marked with DyLight 405 and Rhodamine Red (Jackson ImmunoResearch, West Grove, PA). These were imaged with confocal microscopy using the 405, 488, and 561 laser lines. Widefield fluorescence microscopy was used in some experiments to capture GFP, Alexa 488, or Rhodamine Red fluorescence with Zeiss 45 or Zeiss 49 filters. The number of PGP-2 marked organelles in individual embryos was quantified using SQUASSH software analysis of confocal Zstacks spanning the entire intestine [64]. Using confocal Z-stacks, the diameter of CDF-2::GFP or anti-LMP-1 marked organelles was determined using Zeiss Zen Blue 2012 software. In glo-3 (kx38) containing strains, only the diameter of CDF-2::GFP organelles that also contained PGP-2 were measured, as CDF-2::GFP was mislocalized to non-PGP-2 containing lysosomes in glo-3(kx38) mutants. To determine endolysosome size, only LMP-1 containing organelles that lacked the gut granule marker PGP-2 were measured.
For colocalization studies, two or three channel confocal Z-stacks were acquired and analyzed. As noted in the figure legends, either manual or automated colocalization scoring was performed. In some experiments, randomly selected organelles labeled by one marker were manually scored for the presence of a second marker. Individual organelle signals were scored as colocalizing if they overlapped by more than 50%. The colocalization per embryo was calculated and these values were used to determine the mean colocalization shown in the graphs. In other experiments, SQUASSH software was used to segment the Z-stack and every identified organelle was used in the analysis (typically 120-160 gut granules per wild-type embryo). The resulting C size measurement of colocalization represents the fraction of the total volume of one marker that overlapped with the second marker [64]. For example, C size (PGP-2 LMP-1 /PGP-2) refers to the area of PGP-2 that overlapped with LMP-1 divided by the total area of PGP-2, and represents the proportion of PGP-2 that colocalizes with LMP-1. The C size per embryo was calculated and these values were used to determine the mean colocalization shown in the graphs.
One way ANOVAs were carried out in Microsoft Excel for Mac 2011. Bonferroni or Tukey-Kramer post hoc tests were used when making 3 or more comparisons. Bar graphs were generated with Excel for Mac 2011 and dot plots were made with R (version 3.1.2) Beeswarm (Version 0.1.6). Figures were constructed with Photoshop CS2 and representative images used to determine marker colocalization and organelle presence, number, or size is shown. Brightness and contrast adjustments were uniformly applied to each panel.

Yeast 2-hybrid assays
The S. cerevisiae EGY48 strain was used for all 2-hydrid assays [128]. The DupLEX-A yeast 2-hybrid system was used according to the manufacturer's instructions (Origene Technologies, Rockville, MD, USA). The bait plasmids pEG202 and pEG202-NLS encoding LexA DNA binding domain fusions and prey plasmid pJG4-5 encoding B42 transcription activation domain fusions were used. A full-length glo-3 cDNA was PCR amplified from pDONR/Zeo-glo-3 using (italics are homologous to vector sequences and bold hybridize with the coding sequence) P1129 5'CAGATTATGCCTCTCCCGCCATGTTTGGTTATGTTGTTGTTAAT GAAC3' and P1130 5'GCGAAGAAGTCCAAAGCTTCGGTTATTTTAACTGTTTTAAC ACGCATTCC3' with Q5 High Fidelity DNA polymerase (NEB, Ipswich, MA, USA) and inserted into pJG4-5 digested with EcoRI and XhoI using NEBuilder HiFi DNA Assembly Cloning as described by the manufacturer (NEB). A full-length ccz-1 cDNA was amplified from pDONR/Zeo-ccz-1 using P1119 'AACGGCGACTGGCTGGCCATGGAGTCGATTGC AAATCCATTG3' and P1120 5'CTTGGCTGCAGGTCGACGGTCAACTAAAAAATAT GGCTTCGAAATGGG3' with Q5 High Fidelity DNA polymerase (NEB) and inserted into pEG202 digested with EcoRI and XhoI, using NEBuilder HiFi DNA Assembly Cloning as described by the manufacturer (NEB). Sequencing of the resulting plasmids showed that the coding sequences lacked mutations and were in-frame with the DNA binding or transcription activation domains (Genewiz, South Plainfield, NJ, USA). Lithium acetate mediated transformation was used to simultaneously introduce combinations of plasmids into EGY48. LEU2 reporter expression was assessed by growing strains in 2% dextrose lacking histidine, tryptophan, and uracil liquid media overnight, diluting to 1.0 OD600, and spotting this and serial dilutions on 2% dextrose or 2% galactose/1% raffinose plates lacking leucine, histidine, tryptophan, and uracil at 30˚C for 3 days. The pSH18-34 reporter plasmid was used to assess lacZ expression by growing strains on 2% dextrose or 2% galactose/1% raffinose plates containing 80μg/ml X-Gal and lacking histidine, tryptophan, and uracil at 30˚C for 3 days.
(A and C) Signals from antibodies to PGP-2 and RAB-7 or LMP-1 were acquired in 1.5-fold stage embryos using confocal microscopy. (A) In wild type and tbc-2(tm2241), RAB-7 did not associate with gut granules (white arrows in insets). (C) PGP-2 compartments contained LMP-1 in wild type and tbc-2(tm2241) mutants (black arrows in insets). (B and D) Colocalization was scored by randomly selecting 20-30 PGP-2 marked organelles in five 1.5-fold stage embryos and assessing the presence of RAB-7 or LMP-1 signals. Five embryos were scored for each genotype. The mean is plotted and bars represent the 95% confidence interval. �� indicates p�0.001 by one way ANOVA comparing tbc-2(tm2241) to wild type. (TIF) S1 Supporting Numerical Data. This spreadsheet contains all of the numerical data that underlies the graphs in the manuscript. (XLSX)