C. elegans intestinal cells are characterized by the presence of gut granules, lysosome-related organelles (LROs) that contain autofluorescent and birefringent material. Gut granule biogenesis requires the activity of evolutionarily conserved genes that function in directing and transporting proteins to LROs. There are likely many undiscovered genes necessary for the generation of LROs. To identify these genes, we have screened 1336 Million Mutation Project (MMP) strains and found 137 strains with altered gut granule formation, size, and/or positioning. We identified 78 MMP strains with altered gut granule biogenesis. Seven strains have nonsense mutations likely to disrupt the function of 5 genes known to function in gut granule formation. 23 strains have missense mutations in 10 genes that act in gut granule formation. The 48 remaining strains lack mutations in known gut granule biogenesis genes. In 21 of these strains, we have characterized multiple alleles and used rescue experiments to identify mutations in
glo-9,
glo-10,
lyst-1, and
wht-2 as causing the defects in gut granule formation. These four genes represent new factors promoting gut granule biogenesis. We are continuing experiments to identify the genes causing altered gut granule formation in the 27 remaining strains. 90 of the MMP strains have increased or decreased gut granule size. Four of these strains have nonsense mutations likely to disrupt function of 2 genes known to play a role in gut granule morphology. 13 strains have missense mutations in 9 genes known to play a role in both gut granule morphology and biogenesis. In 11 strains we have used multiple alleles to identify mutations in
glo-9 and
glo-10 as causing alterations in gut granule morphology. We are continuing our analyses of the remaining 62 strains. We identified two strains with altered gut granule positioning and have used rescue experiments to show that they result from mutations in
wht-2 and
wht-7. Here we present our analyses of
glo-9, which encodes a regulatory protein that likely impacts Arf GTPase activity.
glo-9 mutants display enlarged gut granules that mislocalize the gut granule proteins LMP-1 and CDF-2::GFP. Introduction of N- or C-terminally GFP tagged forms of GLO-9 rescue these phenotypes. Introduction of GLO-9 containing a point mutation predicted to alter its effects on Arf activity no longer rescues, consistent with GLO-9 acting as an Arf regulator in gut granule formation. The GFP tagged forms of GLO-9 localize to the Golgi, which is often closely associated with gut granules. Together this work implicates Arf mediated trafficking between the Golgi and gut granules in LRO biogenesis.