Progress 08/15/08 to 08/14/10
Outputs
OUTPUTS: Through this work, we have begun to decipher how peroxisomes dispose of damaged or no longer needed proteins. This work has been disseminated to scientists and students in seminars given by Bonnie Bartel at the Buenos Aires Plant Biology Lectures at the Universidad de Buenos Aires in Argentina on October 28, 2008; at the Department of Plant Biology of Cornell University in Ithaca, NY on September 26, 2008; and at the Molecular and Environmental Plant Sciences (MEPS) Symposium at Texas A&M University in College Station, TX on March 4, 2009. PARTICIPANTS: Postdoctoral fellow Matthew J. Lingard was supported by this grant and worked on the aims of the grant from 8/15/08 until he began his job at Monsanto in March of 2009. Bonnie Bartel did not receive support from the grant, but advised Dr. Lingard in his experiments, assisted in designing experiments and interpreting results, and participated in preparing the data for publication. TARGET AUDIENCES: Dr. Lingard served as a research mentor to a Rice University undergraduate, Chaya Murali, who assisted with the experiments described in aim 1. Ms. Murali was trained in plant molecular genetics techniques, and is continuing the experiments of aim 1. She provides weekly email reports of her progress and will be presenting a poster at the Rice Undergraduate Research Symposium in the spring of 2009. PROJECT MODIFICATIONS: Dr. Lingard has accepted a job at Monsanto less than a year into the 2-year funding period. Because of this abbreviated effort, not all of the proposed experiments were completed. For aim 1, identifying and characterizing GFP-ICL degradation signals, all of the constructs were made and transformed into plants, and homozygous progeny lines have been obtained for most constructs. We are in the process of monitoring stability of the reporter proteins. For aim 2, reverse genetic screens for mutants with delayed ICL and MLS degradation, many of the mutants in table 1 have been analyzed. Analysis of the peroxin mutants is described in our Lingard et al. 2009 PNAS paper. In addition, all of the confirmed peroxisomal proteases with available insertion lines have been assayed for ICL and MLS degradation, and no defects were observed. However, one of these protease mutants does display defects in peroxisomal matrix protein targeting, and we currently are preparing a manuscript for publication describing these defects. For aim 3, developing rice as a new model for peroxisome biogenesis and function, we have obtained several putative insertional alleles disrupted in rice peroxins. We have grown up the initial seed, confirmed genotypes, and are awaiting the next generation for phenotypic analyses.
Impacts
Peroxisomes are ubiquitous eukaryotic organelles housing diverse enzymatic reactions, including several that produce toxic reactive oxygen species. Although understanding of the mechanisms whereby enzymes enter peroxisomes with the help of peroxin (PEX) proteins is increasing, mechanisms by which damaged or obsolete peroxisomal proteins are degraded are not understood. We have exploited unique aspects of plant development to characterize peroxisome-associated protein degradation (PexAD) in Arabidopsis. Oilseed seedlings undergo a developmentally regulated remodeling of peroxisomal matrix protein composition in which the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS) are replaced by photorespiration enzymes. We found that mutations expected to increase or decrease peroxisomal H2O2 levels accelerated or delayed ICL and MLS disappearance, respectively, suggesting that oxidative damage promotes peroxisomal protein degradation. ICL, MLS, and the beta-oxidation enzyme thiolase were stabilized in the pex4-1 pex22 1 double mutant, which is defective in a peroxisome-associated ubiquitin-conjugating enzyme and its membrane tether. Moreover, the stabilized ICL, thiolase, and an ICL-GFP reporter remained peroxisome associated in pex4-1 pex22-1. ICL also was stabilized and peroxisome associated in pex6-1, a mutant defective in a peroxisome-tethered ATPase. ICL and thiolase were mislocalized to the cytosol but only ICL was stabilized in pex5-10, a mutant defective in a matrix protein import receptor, suggesting that peroxisome entry is necessary for degradation of certain matrix proteins. Together, our data reveal new roles for PEX4, PEX5, PEX6, and PEX22 in PexAD of damaged or obsolete matrix proteins in addition to their canonical roles in peroxisome biogenesis.
Publications
- Lingard, M.J., Monroe-Augustus, M., and Bartel, B. (2009) Peroxisome-associated matrix protein degradation in Arabidopsis. Proc. Natl. Acad. Sci. USA 106, 4561-4566.
|