Graduate and Postdoctoral Studies
BioSciences-Biochemistry and Cell Biology
Insights from forward- and chemical-genetic studies on peroxisome functions, peroxisome-environment interactions, and the peroxisome-associated ubiquitination.
Wednesday, April 12, 2017
to 4:00 PM
102 Keck Hall
Subcellular compartments (organelles) enclose metabolic pathways to increase enzyme-substrate interactions and prevent leakage of harmful byproducts. The peroxisome is an organelle housing many critical oxidative reactions. In humans, a suite of genetic peroxisome biogenesis disorders affect approximately 1 in 50,000 births in North America. No effective treatments are available, and these disorders often result in death during infancy. In oilseed plants, such as Arabidopsis thaliana, peroxisomes metabolize stored fatty acids to fuel germination and early development. Peroxisomes are the sole site of fatty acid ?-oxidation in plants, and as in mammals, complete peroxisome dysfunction confers lethality. Although a general framework for understanding peroxisome biogenesis is in place, detailed biogenesis mechanisms remain unclear and how peroxisomes respond to environmental challenges is not well understood.
Proteins required for peroxisome biogenesis or protein import into the organelle are named peroxins or PEX proteins. Peroxisomal matrix proteins carry peroxisome-targeting signals that are bound by the receptors PEX5 and PEX7. Cargo-receptor complexes dock on the peroxisomal membrane and deliver cargo into the organelle. PEX5 is a shuttling receptor, traveling between the peroxisome and the cytosol. Peroxisome-associated ubiquitination enzymes singly or multiply ubiquitinate PEX5 to signal either return to the cytosol via a peroxisome-tethered ATPase complex or degradation by the proteasome, respectively. The peroxisome-associated ubiquitination machinery includes a ubiquitin-conjugating enzyme (PEX4) and three RING ubiquitin-protein ligases (PEX2, PEX10, and PEX12). I used mutants with defective peroxisome-associated ubiquitination machinery to dissect peroxin functions. I found that RING peroxin mutants displayed elevated levels of peroxisomal cargo receptors, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Disruption of any Arabidopsis RING peroxin decreased PEX10 levels, indicating that each RING peroxin is required for RING complex stability, as in yeast and mammals. Moreover, reducing PEX4 function in the pex12-1 mutant restored PEX10 levels and partially ameliorated molecular and physiological defects, leading to the hypothesis that PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue destabilizes the RING peroxin complex in pex12-1.
Peroxisomes are dynamic; peroxisomal contents and abundance change to accommodate developmental stages and environmental stimuli, including salinity and high temperature. I found that although growth on high salt promotes peroxisome proliferation, levels of certain peroxisomal proteins were reduced. Comparison of salt responses of various mutants revealed that salt-induced ?-oxidation-dependent oxidative stress promoted peroxisomal protein degradation and that catalase protected peroxisomal proteins from degradation.
I found that mildly elevated growth temperature alleviated peroxisomal defects in the ubiquitin-conjugating enzyme mutant pex4-1. The slowed ubiquitination of the cargo receptor PEX5 in pex4-1 increases PEX5 membrane association, and growth at elevated temperature increased proteasomal degradation of PEX5 to reduce overall PEX5 levels. My results support the hypothesis that efficient retrotranslocation of PEX5 after cargo delivery is needed not only to make PEX5 available for further rounds of cargo delivery, but also to prevent the peroxisome dysfunction that results from PEX5 lingering in the peroxisomal membrane.
Lastly, I developed a chemical-genetic approach to identify several small molecules that disrupt peroxisome functions. One lead compound conferred ?-oxidation deficiency and decreased levels of docking peroxins, PEX13 and PEX14. This lead compound may increase phosphorylation of PEX13, which was likely targeted for degradation. Another lead compound also conferred ?-oxidation deficiency, but the chemical target remains unclear. Forward-genetic screens coupled with whole-genome sequencing to identify potential targets of this compound are ongoing.
Overall, this work has increased understanding of individual peroxins and provided insights into peroxisome-environment interactions. The novel chemical-genetic screen identified effective chemical modulators of peroxisome functions. Because peroxisome functions and peroxins are highly conserved, these insights and chemical modulators might be useful in peroxisome studies in diverse organisms.