Using forward genetics to elucidate peroxisome biogenesis and peroxisome-associated protein degradation in Arabidopsis thaliana
Doctor of Philosophy
Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H2O2, which can damage proteins. Little is known about how damaged or obsolete peroxisomal proteins are degraded. Developmentally timed peroxisomal content remodeling in Arabidopsis thaliana provides model substrates with which peroxisome-associated protein degradation can be elucidated. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, seeds expressing GFP-ICL, which is degraded similarly to endogenous ICL, were mutagenized and persistent GFP-ICL fluorescence (pfl) mutants were identified. I have identified the defective genes in 17 pfl mutants. One pfl mutant is missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, one pfl mutant has a novel lesion in the MULTIFUNCTIONAL PROTEIN (MFP/At3g06860) gene, three pfl mutants have mutations in a peroxisomal GLUTAMATE:GLYOXYLATE AMINOTRANSFERASE (GGT1/At1g23310) gene that results in truncated proteins, and two pfl mutants novel mutations in a PEROXISOMAL NAD+ TRANSPORTER (PXN/At2g39970) gene. Four pfl mutants are defective in autophagy-related genes, ATG2/At3g19190 and ATG7/At5g45900. Three pfl mutants are defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix. One pfl mutant displays normal matrix protein import carries a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. Two pfl mutants are defective in peroxisomal membrane proteins containing RING-finger domains, PEROXIN2 (PEX2/At1g79810) and PEROXIN10 (PEX10/At2g26350). The pfl screen has provided a rich source for novel peroxisome-defective mutants, including several that have not emerged from previous screens. Characterization of these pfl mutants suggests that peroxisomal metabolism influences the rate of matrix protein degradation. The identification of pex14 mutants indicates peroxisomal matrix proteins must enter the peroxisome for efficient degradation, and the isolation of pex2-1, pex6-2, and pex10-2 as pfl mutants supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation. Beyond insights into the mechanisms of peroxisome-associated protein degradation, the pfl mutants have provided new tools for elucidating the components and mechanisms of peroxisome biogenesis in plants.
Peroxisome remodeling; Peroxin; Arabidopsis thaliana; Protein degradation