Peroxisomal function is critical for a number of metabolic pathways including the beta-oxidation of very long chain fatty acids and the synthesis of glycerolipids. Disruption of any of the genes required for peroxisome biogenesis results in a block of all peroxisomal metabolic functions. Defects in peroxisome biogenesis genes in humans cause a group of disorders known collectively as the peroxisome biogenesis disorders. Previous studies using injection of dsRNA to disrupt peroxisome biogenesis genes in C. elegans resulted in a developmental arrest of the F1 generation at the L1/L2 stage (1,2). Targeting the same peroxisomal biogenesis genes with the feeding RNAi method, a previously unreported phenotype was found in the parental adult animals. The parental animals grow at a normal rate but develop large inclusion bodies in intestinal cells 4-5 days after becoming gravid adults. The inclusion bodies resemble lipid droplets but do not stain with the vital dye Nile Red. To gain a better understanding of the mechanisms underlying both the F1 developmental arrest and the adult intestinal inclusion body phenotypes we undertook a screen to examine the RNAi phenotype of each individual peroxisomal enzyme. Proteins that participate in peroxisomal metabolic pathways are imported from the cytosol into the peroxisomal matrix in a process involving a specific targeting signal. The type 1 peroxisomal targeting signal (PTS1) used by C. elegans consists of a tripeptide located at the C-terminus of the protein (S/A/C-K/R/H-L/M/I). The tripeptide SKL is most often used, however conserved variants are also found. Over 100 C. elegans proteins that contain a putative peroxisomal targeting signal were identified and tested for their RNAi phenotype. The screen was performed in the sensitized background of
rrf-3 animals and the bacteria were induced with lactose instead of IPTG to achieve a more robust knockdown. RNAi of two peroxisomal genes, 3 ketoacyl thiolase, and
dhs-28, resulted in the adult intestinal inclusion body phenotype. Both of these proteins function in the beta-oxidation of very long chain fatty acids. RNAi of both of these genes as well as 5 others results in slowed growth or arrest at the L1/L2 stage in the F1 generation. Three of these function as enzymes in the beta-oxidation pathway and the other 2 are required for ether lipid biosynthesis. (1) Petriv OI, Pilgrim DB, Rachubinski RA, Titorenko VI (2002). Physiol Genomics 10:79-91. (2) Thieringer H, Moellers B, Dodt G, Kunau WH, Driscoll M (2003). J Cell Science 16: 1797-1804.