Function of theCaenorhabditis elegansABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle

Abstract

Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(-) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase-containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(-) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red-stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red-stained fat in pgp-2(-) animals as well as insight into the specialized function of this lysosome-related organelle.

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans

“…The discrepancy between the Nile red staining and the three other methods used to monitor lipid stores, i.e., CARS microscopy, Sudan black, and gas chromatography, point to some limitation of the Nile red staining method as an indicator of lipid store status in C. elegans. Indeed, others have shown that Nile red merely stains lysosome-related compartments in C. elegans (22,37), and our results also show that Nile red allows the visualization of only a subset of the lipid droplets. There are obviously a variety of gut granules types in the C. elegans intestine, of which only a subset is stained by Nile red, and staining of this subset by Nile red does not always correlate with lipid content.…”

“…To monitor protein prenylation in C. elegans, we adapted a GFP-based method previously described for mammalian cells (41). The plasmid pGLO-1P::GFP-CAAX carries the intestinal-specific promoter glo-1 (22,42) to express a modified GFP fused to the last 12 aa of the C. elegans ras-2 gene, including the terminal prenylation motif sequence CLIS (see SI Materials and Methods). pGLO-1P::GFP-CAAX was microinjected together with the phenotypic marker rol-6 carried on plasmid pRF4 (43), and transgenic worms were maintained by picking individuals with the roller phenotype.…”

“…S3). Schroeder and coworkers have previously shown that Nile red stains lysosome-like granules rather than lipid droplets within the C. elegans intestine (22). However, our efforts to document defects in lysosomes has so far been negative (Fig.…”

A Regulatory Module Controlling Pharyngeal Development and Function in Caenorhabditis elegans

“…These observations, together with our observation that sup-37 is expressed strongly in the larval pharynx (see below), suggested that sup-37 null mutants may have defective pharynges that preclude proper feeding. Consistent with a feeding defect, the intestines of the mutants have refractile vacuoles indicative of starvation (Schroeder et al 2007). To test this, homozygous tm356 and tm1083 mutants were fed fluorescent Escherichia coli (OP50) and analyzed by compound microscopy.…”

The Intestinal Copper Exporter CUA-1 Is Required for Systemic Copper Homeostasis in Caenorhabditis elegans

“…Upon RNAi knockdown of cua-1, the number and morphology of LysoTracker-positive compartments did not change. However, RNAi knockdown pgp-2, which encodes an ABC transporter that is required for gut granule biogenesis and localizes to the gut granule membrane (35,37), significantly reduced LysoTracker staining and prevented CUA-1.1::GFP-containing vesicle formation, whereas CUA-1.1::GFP was detected on the basolateral membrane (Fig. 6A).…”