Lysosome-related organelles (LROs) comprise a class of cell-type specific compartments with specialized functions. In mammals, defects in their formation result in Hermansky-Pudlak syndrome. The genes controlling the biogenesis of LROs are conserved in metazoa and include subunits of the AP-3 and HOPS complexes and Rab32/38. GLO-1, a homologue of Rab32/38, is localized to and is required for the formation of C. elegans gut granules, LROs containing fat and birefringent material found only within intestinal cells. In an effort to better understand the activity of GLO-1, we have carried out a screen for mutations the suppress gut granule biogenesis defects associated with the loss of
glo-1 function. To date, we have molecularly characterized 16 mutant alleles of
glo-1: 6 are missense alleles altering amino acid residues conserved in all Rab GTPase, 3 are amber nonsense alleles, 2 are opal nonsense alleles, 2 are alleles predicted to alter splicing, and 2 are alleles that delete the entire
glo-1 gene. All of the alleles result in the complete loss of birefringent material from embryonic intestinal cells and its mislocalization into the embryonic intestinal lumen. In order to identify both bypass and allele specific suppressors we screened for suppressors of
glo-1(
kx8), an allele that changes Asp67 to Asn. In other small GTPases, this residue has been shown to be essential for their function. We carried out a screen of F2 embryos within approximately 100,000 mutagenized
glo-1(
kx8) F1 adults for the presence of birefringent compartments within their intestinal cells. From the screen we identified 8
glo-1 suppressors (
kx101-
kx108) that fall into 2 distinct classes. Six of the alleles are recessive and weakly restore birefringent compartments in
glo-1(
kx8) embryos. However, other markers of gut granules do not appear to be similarly restored, suggesting that these suppressors alter the trafficking or formation of the birefringent material. Two suppressors,
kx105 and
kx106, dominantly and with high penetrance, suppress the loss and mislocalization of birefringent material. Suppressed embryos contain organelles that exhibit all of the hallmarks of normal gut granules.
kx105 suppresses the loss of gut granules in multiple
glo-1 alleles, including those containing very early premature stop codons, indicating that it bypasses the function of
glo-1 in gut granule biogenesis. We are currently investigating whether the dominant suppressors can similarly suppress the defects in gut granule biogenesis exhibited by AP-3 and HOPS mutants. The dominant suppressors are not present on the X chromosome where
glo-1 is located, and we are carrying out assays to identify the gene(s) altered by these alleles.