Browsing by Author "Rinaldi, Mauro Adriel"

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    Insight into peroxisome biogenesis and metabolism from a microscopy-based screen for Arabidopsis thaliana mutants with deviations in peroxisomal morphology, distribution, or import
    (2016-04-20) Rinaldi, Mauro Adriel; Bartel, Bonnie; Bennett, George N; Raphael, Robert
    Key steps of vital metabolic pathways are housed in peroxisomes, essential organelles. Intensive research has advanced our understanding of peroxisomes, but there are still gaps in our models. For example, we do not fully understand the additional roles of β-oxidation and peroxisomal cofactor homeostasis. Therefore, I conducted a microscopy-based screen for aberrant distribution of peroxisomally-targeted fluorescence in Arabidopsis thaliana. This screen uncovered novel alleles defective in lipid body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, auxin metabolism, pexophagy and other peroxisomal processes. Weak mutants in lipid mobilization retained lipid bodies even without displaying other defects, suggesting that microscopy was sensitive to small deficiencies and that fatty acid β-oxidation happens at higher rates than those needed for normal growth. Moreover, analysis of these mutants revealed that fatty acid β-oxidation was needed for peroxisomal matrix protein import and wild-type peroxisome morphology. Epistasis analysis suggested that CoA and NAD+ import contributes to the peroxisomal pool and to β-oxidation. Mutants defective in PECTIN METHYLESTERASE 31 were also recovered, suggesting a role in lipid mobilization for this cytosolic protein. Peroxisomal processes rely on the action of peroxins (PEX proteins), factors needed to build and maintain peroxisomes. One of the most important peroxins is the ATPase PEX1, which together with the similar ATPase PEX6, recycles the peroxisomal matrix protein receptor PEX5 from the peroxisome membrane following cargo delivery. Mutations in human PEX1 can lead to lethal peroxisomal biogenesis disorders. However, very little is known about the role of PEX1 in plants. My screen yielded a PEX1 mutation that was lethal when homozygous, suggesting PEX1 is needed for peroxisomes and life. pex1 mutants accumulated reduced levels of PEX5 and PEX6, suggesting that PEX1 promotes PEX5 and PEX6 stability. PEX6 overexpression rescued a pex1 mutant. These data suggest that the heterohexamer model of PEX1-PEX6 function developed in other organisms is conserved in plants. Unlike PEX6 overexpression, PEX1 overexpression in Arabidopsis leads to growth defects, suggesting that PEX1 levels must remain within a narrow range. The screen performed for this thesis demonstrates the sustained power of forward genetic screens to uncover new factors in biological processes.