Browsing by Author "Kao, Yun-Ting"

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    A PEX5 missense allele preferentially disrupts PTS1 cargo import into Arabidopsis peroxisomes
    (American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd., 3/20/2019) Patel, Khushali J.; Kao, Yun-Ting; Llinas, Roxanna J.; Bartel, Bonnie; BioSciences
    The sorting of eukaryotic proteins to various organellar destinations requires receptors that recognize cargo protein targeting signals and facilitate transport into the organelle. One such receptor is the peroxin PEX5, which recruits cytosolic cargo carrying a peroxisome‐targeting signal (PTS) type 1 (PTS1) for delivery into the peroxisomal lumen (matrix). In plants and mammals, PEX5 is also indirectly required for peroxisomal import of proteins carrying a PTS2 signal because PEX5 binds the PTS2 receptor, bringing the associated PTS2 cargo to the peroxisome along with PTS1 cargo. Despite PEX5 being the PTS1 cargo receptor, previously identified Arabidopsis pex5 mutants display either impairment of both PTS1 and PTS2 import or defects only in PTS2 import. Here, we report the first Arabidopsis pex5 mutant with an exclusive PTS1 import defect. In addition to markedly diminished GFP‐PTS1 import and decreased pex5‐2 protein accumulation, this pex5‐2 mutant shows typical peroxisome‐related defects, including inefficient β‐oxidation and reduced growth. Growth at reduced or elevated temperatures ameliorated or exacerbated pex5‐2 peroxisome‐related defects, respectively, without markedly changing pex5‐2 protein levels. In contrast to the diminished PTS1 import, PTS2 processing was only slightly impaired and PTS2‐GFP import appeared normal in pex5‐2. This finding suggests that even minor peroxisomal localization of the PTS1 protein DEG15, the PTS2‐processing protease, is sufficient to maintain robust PTS2 processing.
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    Disparate peroxisome‐related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization
    (Wiley, 2017) Gonzalez, Kim L.; Fleming, Wendell A.; Kao, Yun-Ting; Wright, Zachary J.; Venkova, Savina V.; Ventura, Meredith J.; Bartel, Bonnie
    Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail‐anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β‐oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome‐associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.
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    Elevated growth temperature decreases levels of the PEX5 peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4 ubiquitin-conjugating enzyme
    (BioMed Central, 2015) Kao, Yun-Ting; Bartel, Bonnie
    Background: Peroxisomes house critical metabolic reactions. For example, fatty acid β-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants β-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. Results: We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. Conclusions: We conclude that growth at elevated temperature increases proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. Our 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.
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    Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components
    (American Society of Plant Biologists, 2016) Kao, Yun-Ting; Fleming, Wendell A.; Ventura, Meredith J.; Bartel, Bonnie; Biochemistry and Cell Biology Program
    Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.
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    Insights from forward- and chemical-genetic studies on peroxisome functions, peroxisome-environment interactions, and the peroxisome-associated ubiquitination
    (2017-04-20) Kao, Yun-Ting; Bartel, Bonnie
    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 oilseed plants, such as Arabidopsis thaliana, peroxisomes metabolize stored fatty acids to fuel germination and early development. 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. PEX5 is a shuttling peroxisomal matrix protein receptor, traveling between the peroxisome and the cytosol. Peroxisome-associated enzymes ubiquitinate PEX5 to signal either return to the cytosol or degradation by the proteasome. I characterized mutants with defective peroxisome-associated ubiquitination machinery and found that mutations in the peroxisome-associated ubiquitin-conjugating enzyme (PEX4) and three RING ubiquitin-protein ligases (PEX2, PEX10, and PEX12) conferred elevated PEX5 levels. 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 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, presumably due to β-oxidation-dependent oxidative stress promoted peroxisomal protein degradation and that catalase protected peroxisomal proteins from degradation. I also found that mildly elevated growth temperature increased proteasomal degradation of PEX5 to reduce overall and detrimental membrane-associated PEX5 levels and alleviated peroxisomal defects in the ubiquitin-conjugating enzyme mutant pex4-1. Overall, this work has increased understanding of individual peroxins and provided insights into peroxisome-environment interactions. I also developed chemical-genetic screens to identify 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.
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    Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis
    (American Society of Plant Biologists, 2014) Burkhart, Sarah E.; Kao, Yun-Ting; Bartel, Bonnie
    Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.
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    Peroxisome Function, Biogenesis, and Dynamics in Plants
    (American Society of Plant Biologists, 2018) Kao, Yun-Ting; Gonzalez, Kim L.; Bartel, Bonnie
    Eukaryotic cells employ organellar compartmentalization to increase efficiency of cellular processes and protect cellular components from harmful products, such as reactive oxygen species. Peroxisomes are organelles that sequester diverse oxidative reactions and play important roles in metabolism, reactive oxygen species detoxification, and signaling. Oxidative pathways housed in peroxisomes include fatty acid β-oxidation, which contributes to embryogenesis, seedling growth, and stomatal opening. Other peroxisomal enzymes enable photorespiration, which increases photosynthetic efficiency. Peroxisomes contribute to the synthesis of critical signaling molecules including the jasmonic acid, auxin, and salicylic acid phytohormones. Peroxisomes lack DNA; peroxisomal proteins are encoded in nuclear DNA and posttranslationally enter the organelle. Recent studies have begun to fill gaps in our understanding of how peroxisomal proteins are imported, regulated, and degraded. Despite this progress, much remains to be learned about how peroxisomes originate from the ER, divide, and are degraded through pexophagy, a form of organelle-specific autophagy. Peroxisomes play vital roles in multiple aspects of plant life, and in this review, we highlight recent advances in our understanding of plant peroxisome functions, biogenesis, and dynamics, while pointing out areas where additional studies are needed.