Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.     

Peroxisomal ATP Import Is Essential for Seedling Development in Arabidopsis thaliana

Several recent proteomic studies of plant peroxisomes indicate that the peroxisomal matrix harbors multiple ATP-dependent enzymes and chaperones. However, it is unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether external ATP fuels the energy-dependent reactions within peroxisomes. The existence of transport proteins that supply plant peroxisomes with energy for fatty acid oxidation and other ATP-dependent processes has not previously been demonstrated. Here, we describe two Arabidopsis thaliana genes that encode peroxisomal adenine nucleotide carriers, PNC1 and PNC2. Both proteins, when fused to enhanced yellow fluorescent protein, are targeted to peroxisomes. Complementation of a yeast mutant deficient in peroxisomal ATP import and in vitro transport assays using recombinant transporter proteins revealed that PNC1 and PNC2 catalyze the counterexchange of ATP with ADP or AMP. Transgenic Arabidopsis lines repressing both PNC genes were generated using ethanol-inducible RNA interference. A detailed analysis of these plants showed that an impaired peroxisomal ATP import inhibits fatty acid breakdown during early seedling growth and other β-oxidation reactions, such as auxin biosynthesis. We show conclusively that PNC1 and PNC2 are essential for supplying peroxisomes with ATP, indicating that no other ATP generating systems exist inside plant peroxisomes.The β-oxidation of fatty acids, a process that exclusively occurs within peroxisomes in plants and yeast, plays an important role in storage oil mobilization to support seedling establishment of oilseed plants, such as Arabidopsis thaliana (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Upon germination, fatty acids are released from storage oil triacylglycerol (TAG) by lipolysis, degraded via β-oxidation in specialized peroxisomes, termed glyoxysomes, and subsequently converted to sucrose, which drives growth and development until seedlings become photoautotrophic (Graham and Eastmond, 2002; Baker et al., 2006; Graham, 2008). Before the fatty acids can enter β-oxidation, they are imported into peroxisomes by a peroxisomal ATP binding cassette (ABC) transporter, variously known as CTS (COMATOSE), At PXA1 (Arabidopsis peroxisomal ABC transporter), or PED3 (peroxisomal defective 3) and hereafter referred to as CTS (Zolman et al., 2001; Footitt et al., 2002; Hayashi et al., 2002). Subsequently, the imported fatty acids are activated by esterification to CoA. This ATP-dependent reaction within peroxisomes is catalyzed by long-chain acyl-CoA synthetases 6 and 7 (LACS6 and LACS7, respectively), which are named according to their substrate specificity for long-chain fatty acids, which are significant components of seed storage oil in Arabidopsis (Fulda et al., 2002, 2004).In Saccharomyces cerevisiae, two mechanisms exist for import and activation of fatty acids, depending on chain length (Hettema et al., 1996). Long-chain fatty acids (C16 and C18) are converted to acyl-CoA esters in the cytosol prior to transport by the heterodimeric peroxisomal ABC transporter, Pxa1p/Pxa2 (Hettema et al., 1996). By contrast, short- and medium-chain fatty acids (≤C14) that enter the peroxisomes by passive diffusion or by an unknown transport protein are activated within peroxisomes (Hettema et al., 1996). The possibility cannot be excluded, though, that CTS imports the corresponding CoA derivatives, as is the case for the yeast Pxa1p/Pxa2p heterodimer (Hettema et al., 1996; Verleur et al., 1997), implicating a cytosolic activation of the fatty acids, catalyzed by a hitherto unknown enzyme. The actual substrates transported by CTS in Arabidopsis have not yet been experimentally determined (Theodoulou et al., 2006). However, the sucrose-dependent seedling growth phenotype of the lacs6 lacs7 double knockout mutant demonstrated that peroxisomal activation is essential for lipid mobilization to provide energy for early seedling growth (Fulda et al., 2004). The lacs6 lacs7 mutant is impaired in the degradation of fatty acids, leading to growth arrest shortly after germination (Fulda et al., 2004).Besides fatty acid mobilization, β-oxidation is also involved in generation of signaling molecules, such as the phytohormones auxin and fatty acid–derived jasmonic acid (JA) (Zolman et al., 2000; Schaller et al., 2004; Delker et al., 2007). By analogy to fatty acids released from storage oil, the precursors of these signaling molecules require CoA esterification before they can enter β-oxidation (Baker et al., 2006; Goepfert and Poirier, 2007). While the enzymes responsible for ATP-dependent activation of natural auxin (indole butyric acid [IBA]) and proherbicide 2,4-dichlorophenoxybutyric acid (2,4-DB) are currently unknown, several enzymes belonging to the acyl-activating enzyme (AAE) family have been implicated in jasmonate biosynthesis (Schneider et al., 2005; Koo et al., 2006; Kienow et al., 2008). Moreover, several as yet uncharacterized members of the large AAE family carry a putative peroxisome targeting signal (PTS) and thus might be good candidates to activate the additional β-oxidation substrates within peroxisomes (Shockey et al., 2002, 2003).In the case where activation of fatty acids or other substrates takes place within peroxisomes, the question arises as to how these ATP-dependent reactions are supplied with ATP. It is currently unknown whether plant peroxisomes are able to produce ATP by substrate-level phosphorylation or whether they depend on external ATP to supply energy-dependent reactions within peroxisomes. So far, transport proteins that supply plant peroxisomes with energy for fatty acid oxidation have not been characterized. However, in bakers'' yeast, a peroxisomal adenine nucleotide transporter, ANT1, that is required for the ATP-dependent activation of medium-chain fatty acids inside peroxisomes has been characterized (Palmieri et al., 2001).ATP transport proteins play an important role in the distribution of the primary agent coupling endergonic and exergonic reactions in every cellular compartment (Winkler and Neuhaus, 1999). In Arabidopsis and other plants, various adenine nucleotide carriers have been identified at the molecular level. The mitochondrial ADP/ATP carrier mediates the export of ATP that is synthesized in the mitochondrion to provide energy for cellular metabolism (Heimpel et al., 2001; Haferkamp et al., 2002). The plastidial ATP/ADP transporter (nucleotide transporter) is involved in ATP uptake by both chloroplasts and heterotrophic plastids, to enable the nocturnal ATP supply required for chlorophyll biosynthesis (Reiser et al., 2004; Reinhold et al., 2007), as well as by heterotrophic plastids to drive starch biosynthesis (Batz et al., 1992; Tjaden et al., 1998). Yet another ATP/ADP antiporter located in the endoplasmic reticulum (ER) membrane provides energy by importing ATP into the ER for the accumulation of ER-related storage lipids and proteins (Leroch et al., 2008).In this study, we identified two novel peroxisomal adenine nucleotide carrier proteins (PNC1 and PNC2) from Arabidopsis. Colocalization studies demonstrated that these proteins are targeted to peroxisomes. Yeast complementation and in vitro ATP uptake assays showed that both PNC1 and PNC2 catalyze the counterexchange of ATP with AMP. Using an inducible RNA interference (RNAi) repression strategy, we further established several transgenic Arabidopsis lines with reduced expression levels of both PNC1 and PNC2. Our results showed that import of ATP into peroxisomes that is catalyzed by PNC1 and PNC2 is essential for activation of fatty acids during seedling germination and plays a role in other β-oxidation reactions in peroxisomes, such as auxin metabolism. Analysis of PNC1 and PNC2 repression lines further indicates that no other ATP generating systems exist inside plant peroxisomes and that ATP import is the only way to supply the peroxisomal matrix with ATP.     

Pex2 and Pex12 Function as Protein-Ubiquitin Ligases in Peroxisomal Protein Import

The PTS1-dependent peroxisomal matrix protein import is facilitated by the receptor protein Pex5 and can be divided into cargo recognition in the cytosol, membrane docking of the cargo-receptor complex, cargo release, and recycling of the receptor. The final step is controlled by the ubiquitination status of Pex5. While polyubiquitinated Pex5 is degraded by the proteasome, monoubiquitinated Pex5 is destined for a new round of the receptor cycle. Recently, the ubiquitin-conjugating enzymes involved in Pex5 ubiquitination were identified as Ubc4 and Pex4 (Ubc10), whereas the identity of the corresponding protein-ubiquitin ligases remained unknown. Here we report on the identification of the protein-ubiquitin ligases that are responsible for the ubiquitination of the peroxisomal protein import receptor Pex5. It is demonstrated that each of the three RING peroxins Pex2, Pex10, and Pex12 exhibits ubiquitin-protein isopeptide ligase activity. Our results show that Pex2 mediates the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.The maintenance of peroxisome function depends on the formation of the peroxisomal membrane and the subsequent import of both membrane and matrix proteins. Without exception, peroxisomal matrix proteins are nucleus encoded, synthesized on free ribosomes, and subsequently imported in a posttranslational manner (40). The peroxisomal import apparatus can facilitate the transport of folded and oligomeric proteins over the peroxisomal membrane, with the basic principle of this translocation event still being unknown. Based on the concept of cycling receptors (9, 31), the receptor cycle is divided into four steps. In the first step, the cargo proteins are recognized in the cytosol by their cognate receptor protein Pex5 or Pex7. In general, this initial step depends on either one of the two well-characterized PTSs (peroxisomal targeting signals), PTS1 and PTS2, which are recognized and bound by the corresponding receptor proteins Pex5 and Pex7, respectively. In the second step, the cargo-loaded receptors dock with distinct proteins accessible at the surface of the peroxisomal membrane, namely, Pex13 and Pex14. These two proteins together with Pex17 are established components of the docking complex. A second complex of the peroxisomal protein import machinery acts downstream of the docking event and consists of the three peroxins Pex2, Pex10, and Pex12. A common feature of these proteins is a C-terminal RING (really interesting new gene) finger domain. The RING finger subcomplex and the docking subcomplex are both linked in a Pex8-dependent manner to form a larger assembly, the importomer (1). In the third step of the receptor cycle, the cargo is delivered to the peroxisomal matrix, and finally, the receptor is released from the peroxisomal membrane in an ATP-dependent manner and thus made available for proteasomal degradation or another round of import (for a review, see reference 27).With respect to the PTS1 receptor Pex5, recent reports demonstrated that this final ATP-dependent step in the receptor cycle is catalyzed by the AAA (ATPases associated with various cellular activities) peroxins Pex1 and Pex6 (33, 37). The signal for the export process is the attachment of a monoubiquitin moiety or, alternatively, the anchoring of a polyubiquitin chain (5, 35). This protein modification is in general facilitated by a three-step enzyme cascade (20). The ubiquitin (Ub)-activating enzyme (E1) activates the Ub and transfers it to the Ub conjugation enzyme (E2). In a final step, a protein-Ub ligase (E3) binds both E2 and substrate and thereby facilitates the conjugation of the Ub moiety onto the substrate protein. Saccharomyces cerevisiae harbors genes coding for one E1 enzyme, 11 E2 enzymes, and approximately 80 to 100 putative E3 enzymes (18, 29). It was demonstrated that the polyubiquitination of Pex5 primarily depends on the E2 protein Ubc4, which upon deletion can be partly replaced by Ubc5 or Ubc1 (22, 25, 36). Polyubiquitination of Pex5 is not a prerequisite for its function in peroxisomal protein import but might be a crucial step of a quality control system for the disposal of dysfunctional Pex5 (10, 22, 25, 36). Pex5 monoubiquitination is facilitated by the E2 protein Pex4 (Ubc10) in yeast or the Pex4-like UbcH5a/b/c in humans (14, 35, 47). The modification of Pex5 by a single Ub primes the receptor for its export back to the cytosol, where the Ub supposedly is removed prior to the initiation of a new receptor cycle (5, 14, 35). Although the functional relevance and the cognate E2 protein required for the different Ub modifications of Pex5 were identified, the factor(s) determining the substrate specificity, the protein-Ub ligase(s), remained unknown.Here we report on the discovery of the function of Pex2 and Pex12 as E3 proteins required for ubiquitination of the import receptor Pex5. These RING peroxins, defects of which cause the lethal peroxisome biogenesis disorders in humans, exhibit Ub-protein isopeptide ligase activity with Pex5 as the molecular target. Pex2 is shown to mediate the Ubc4-dependent polyubiquitination whereas Pex12 facilitates the Pex4-dependent monoubiquitination of Pex5.     

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins,Is a Redox‐Sensitive Protein

Peroxisome maintenance depends on the import of nuclear‐encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C‐terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N‐terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox‐sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination‐deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.      

Peroxisomal Ubiquitin-Protein Ligases Peroxin2 and Peroxin10 Have Distinct But Synergistic Roles in Matrix Protein Import and Peroxin5 Retrotranslocation in Arabidopsis

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 pex10-2 Arabidopsis 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 pex10-2 double 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.Peroxisomes are dynamic organelles housing critical oxidative metabolic reactions while sequestering harmful reactive oxygen species from the rest of the cell. In Arabidopsis (Arabidopsis thaliana), these single membrane-bound organelles are the sole site of β-oxidation and are essential for normal development (for review, see Hu et al., 2012). Triacylglycerols stored in seeds are cleaved by lipases during germination, and the released fatty acids are β-oxidized in peroxisomes to provide energy for early seedling development (for review, see Graham, 2008). Similarly, an auxin precursor, indole-3-butyric acid (IBA), is β-oxidized to active indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011; Strader et al., 2011). IBA application enhances rooting in many plants (Woodward and Bartel, 2005a), and IAA produced from endogenous IBA promotes hypocotyl elongation, cotyledon expansion, root hair elongation, and lateral root proliferation in Arabidopsis seedlings (Zolman et al., 2001; Strader et al., 2010, 2011; De Rybel et al., 2012).The enzymes needed for β-oxidation and other peroxisomal processes are imported into the peroxisome matrix from their site of synthesis in the cytosol using proteins known as peroxin (PEX) proteins. PEX5 and PEX7 are receptors that recognize peroxisomal targeting signals (PTSs) on proteins destined for the peroxisome matrix. PEX5 recognizes a three-amino acid C-terminal PTS1 (Keller et al., 1987), and PEX7 recognizes a nine-amino acid PTS2 located near the N terminus of the protein (Osumi et al., 1991; Swinkels et al., 1991). In plants, DEG15, a peroxisomal protease, cleaves the N-terminal PTS2 region after the protein enters the peroxisome (Helm et al., 2007; Schuhmann et al., 2008). Cargo-bound PEX5 and PEX7 associate with PEX13 and PEX14 on the peroxisomal membrane (for review, see Azevedo and Schliebs, 2006; Williams and Distel, 2006) and release their cargo into the peroxisome (Fig. 1A). In yeast (Saccharomyces cerevisiae), membrane-associated PEX5 is ubiquitinated, recognized by a complex of ATP-hydrolyzing enzymes comprised of PEX1 and PEX6, and retrotranslocated out of the peroxisome to be used in additional rounds of import (for review, see Fujiki et al., 2012; Grimm et al., 2012).Open in a separate windowFigure 1.Recombination mapping of pfl36 and pfl81 reveals mutations in PEX2 and PEX10. A, PEX proteins (numbered) implicated in matrix protein import serve as receptors (PEX5 and PEX7) recognizing PTS1 or PTS2 cargo proteins, dock receptors at the peroxisomal membrane (PEX13 and PEX14), or assist in PEX5 retrotranslocation (for review, see Hu et al., 2012). In yeast, the RING-finger proteins PEX2, PEX10, and PEX12 participate as heterooligomers in different modes of PEX5 ubiquitination (Ub; for review, see Platta et al., 2013). B, GFP-ICL fluorescence is detected in both 5- and 7-d-old pex10-2 (pfl81) seedlings carrying ICLp:GFP-ICL, whereas GFP-ICL is easily detected in 5- but not 7-d-old wild-type (Wt) ICLp:GFP-ICL seedlings. Hypocotyls of light-grown seedlings were imaged for GFP fluorescence using confocal microscopy. Bar = 20 µm. C, pfl36 was mapped to the bottom of chromosome 1 near the PEX2 gene using the phenotypes of prolonged GFP-ICL fluorescence accompanied by PMDH processing defects. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. D, A gene diagram of PEX2 depicting exons as rectangles and introns as lines. A missense mutation in the fourth exon of PEX2 in pfl36 (pex2-1) changes Arg161 to Lys. Three other pex2 alleles are indicated: pex2-2, ted3 (Hu et al., 2002), and the transfer DNA insertion allele Salk_033081 that confers embryo lethality (Hu et al., 2002). E, The locations of the lesions in viable pex2 alleles are indicated on a diagram depicting the PEX2 protein domains, which include two predicted transmembrane domains (TMDs) and a C-terminal RING domain. F, pfl81 was mapped using the IBA resistance phenotype to an interval on the lower arm of chromosome 2 that contained the PEX10 gene. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. G, pfl81 (pex10-2) carries a PEX10 splicing mutation in the last nucleotide of intron 8. Four other reported pex10 mutants are indicated on the gene diagram: the pex10-1 transfer DNA insertion allele (Schumann et al., 2003; Sparkes et al., 2003) and three Targeting Induced Local Lesions In Genomes (TILLING) alleles: pex10-G93E, pex10-P126S, and pex10-W313* (Prestele et al., 2010). H, The locations of the lesions in the two viable pex10 alleles are indicated on a diagram depicting the PEX10 protein domains, which include two predicted TMDs and a C-terminal RING domain.Yeast PEX5 ubiquitination involves the peroxisomal membrane ubiquitin-protein ligases PEX2, PEX10, and PEX12 (for review, see Platta et al., 2013). The PEX12 ubiquitin-protein ligase monoubiquitinates PEX5 with the assistance of the ubiquitin-conjugating enzyme PEX4 (Platta et al., 2009), allowing PEX5 to be recycled back to the cytosol (Fig. 1A). When PEX4 is absent, yeast ubiquitin-conjugating enzyme4 (Ubc4) works with PEX2 to polyubiquitinate PEX5, marking PEX5 for proteasomal degradation (for review, see Thoms and Erdmann, 2006; Platta et al., 2007, 2013). In yeast, the Really Interesting New Gene (RING) domain of PEX10 binds both PEX2 and PEX12 RING domains to form a trimer (El Magraoui et al., 2012). PEX10 enhances in vitro ubiquitination activity of both PEX2-Ubc4 and PEX12-PEX4 (El Magraoui et al., 2012). Similarly, mammalian PEX12 enhances the in vitro ubiquitination activity of PEX10 (Okumoto et al., 2014). These findings suggest that these RING-finger proteins might act in heteromeric pairs to polyubiquitinate or monoubiquitinate PEX5 (Fig. 1A).Arabidopsis PEX2, PEX10, and PEX12 each display zinc-dependent monoubiquitination activity in vitro (Kaur et al., 2013), but the comparative functions of the Arabidopsis RING-finger PEX proteins in PEX5 ubiquitination, recycling, and degradation have not been reported. This deficiency may, in part, reflect the fact that null alleles of the RING-finger PEX genes confer embryo lethality (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010). RNA interference (RNAi) lines targeting PEX2, PEX10, or PEX12 inefficiently import matrix proteins, display the Suc dependence phenotype that typically accompanies fatty acid β-oxidation defects, and are resistant to 2,4-dichlorophenoxybutyric acid (Fan et al., 2005; Nito et al., 2007), a synthetic analog of IBA (Hayashi et al., 1998). Mutation of any one RING-finger PEX gene results in disassociation or reduced levels of the PEX2-PEX10-PEX12 complex in yeast (Hazra et al., 2002; Agne et al., 2003) and mammals (Okumoto et al., 2014). It is not known whether the defects of the Arabidopsis null and RNAi lines result directly from the loss of catalytic activity of the corresponding RING-finger protein or indirectly from destabilization of the complex and consequent loss of activity of one or both of the associated RING-finger PEX proteins.Only one mutant defective in a RING-finger PEX gene has emerged from forward genetic screens for peroxisome defects in Arabidopsis. A partial loss-of-function pex12 missense allele, aberrant peroxisome morphology4 (apm4), was isolated from a GFP-PTS1 mislocalization screen (Mano et al., 2006). In addition to partially cytosolic GFP-PTS1, apm4 displays a PTS2 processing defect, Suc dependence, and 2,4-dichlorophenoxybutyric acid resistance (Mano et al., 2006), suggesting that PEX12 facilitates peroxisome protein import.In addition to roles in matrix protein import suggested by analysis of RNAi lines (Nito et al., 2007), PEX2 and PEX10 may have plant-specific roles. A pex2 missense allele, ted3, was identified as a dominant suppressor of the photomorphogenic defects of the de-etiolated1 mutant and carries a mutation near the PEX2 RING-finger domain (Fig. 1, D and E; Supplemental Fig. S1; Hu et al., 2002). Moreover, PEX10 RNAi lines display pleiotropic phenotypes not commonly found in Arabidopsis pex mutants, including variegated leaves, reduced fertility (Nito et al., 2007), organ fusions, reduced cuticular wax deposition, and changes in endoplasmic reticulum structure (Kamigaki et al., 2009). Three pex10 alleles generated by TILLING have been reported (Fig. 1G; Supplemental Fig. S2): pex10-W313* truncates the RING-finger domain and is embryo lethal, pex10-G93E germinates but displays seedling lethality, and pex10-P126S displays reduced growth in both normal air and high CO₂ conditions (Prestele et al., 2010). Although GFP-PTS1 is localized in peroxisomes in the pex10-P126S mutant, Suc dependence and IBA resistance were not reported (Prestele et al., 2010).The consequences of overexpressing a mutant version of PEX10 carrying missense mutations in the RING-finger domain (ΔZn) in wild-type Arabidopsis also hint at plant-specific roles for PEX10. PEX10-ΔZn expression confers pleiotropic phenotypes, including smaller cells, serrated leaves, inefficient photorespiration, abnormal peroxisome size and shape, and reduced peroxisome-chloroplast association (Prestele et al., 2010). However, PEX10-ΔZn plants respond like the wild type to IBA and do not require Suc, suggesting that peroxisome metabolism is not dramatically impaired (Schumann et al., 2007; Prestele et al., 2010). In contrast, expressing PEX2-ΔZn in the wild type impairs GFP-PTS1 import without conferring morphological defects, and expressing PEX12-ΔZn confers no abnormal phenotypes (Prestele et al., 2010).Here, we describe the identification and characterization of two unique mutants carrying lesions in Arabidopsis RING-finger PEX genes. We isolated pex2-1 and pex10-2 in a forward genetic screen for genes promoting peroxisomal matrix protein degradation and used these mutants to explore the roles of the corresponding proteins in peroxisome function. We found that PEX2 and PEX10 have independent but related functions that together support PEX5 recycling and matrix protein import into plant peroxisomes.     

The Cytosolic DnaJ-like Protein Djp1p Is Involved Specifically in Peroxisomal Protein Import

The Saccharomyces cerevisiae DJP1 gene encodes a cytosolic protein homologous to Escherichia coli DnaJ. DnaJ homologues act in conjunction with molecular chaperones of the Hsp70 protein family in a variety of cellular processes. Cells with a DJP1 gene deletion are viable and exhibit a novel phenotype among cytosolic J-protein mutants in that they have a specific impairment of only one organelle, the peroxisome. The phenotype was also unique among peroxisome assembly mutants: peroxisomal matrix proteins were mislocalized to the cytoplasm to a varying extent, and peroxisomal structures failed to grow to full size and exhibited a broad range of buoyant densities. Import of marker proteins for the endoplasmic reticulum, nucleus, and mitochondria was normal. Furthermore, the metabolic adaptation to a change in carbon source, a complex multistep process, was unaffected in a DJP1 gene deletion mutant. We conclude that Djp1p is specifically required for peroxisomal protein import.     

A Mobile PTS2 Receptor for Peroxisomal Protein Import in Pichia pastoris

Abstract. Using a new screening procedure for the isolation of peroxisomal import mutants in Pichia pastoris, we have isolated a mutant (pex7) that is specifically disturbed in the peroxisomal import of proteins containing a peroxisomal targeting signal type II (PTS2). Like its Saccharomyces cerevisiae homologue, PpPex7p interacted with the PTS2 in the two-hybrid system, suggesting that Pex7p functions as a receptor. The pex7Δ mutant was not impaired for growth on methanol, indicating that there are no PTS2-containing enzymes involved in peroxisomal methanol metabolism. In contrast, pex7Δ cells failed to grow on oleate, but growth on oleate could be partially restored by expressing thiolase (a PTS2-containing enzyme) fused to the PTS1. Because the subcellular location and mechanism of action of this protein are controversial, we used various methods to demonstrate that Pex7p is both cytosolic and intraperoxisomal. This suggests that Pex7p functions as a mobile receptor, shuttling PTS2-containing proteins from the cytosol to the peroxisomes. In addition, we used PpPex7p as a model protein to understand the effect of the Pex7p mutations found in human patients with rhizomelic chondrodysplasia punctata. The corresponding PpPex7p mutant proteins were stably expressed in P. pastoris, but they failed to complement the pex7Δ mutant and were impaired in binding to the PTS2 sequence.     

SAICAR Induces Protein Kinase Activity of PKM2 that Is Necessary for Sustained Proliferative Signaling of Cancer Cells

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Competitive Microtubule Binding of PEX14 Coordinates Peroxisomal Protein Import and Motility

Human PEX14 plays a dual role as docking protein in peroxisomal protein import and as peroxisomal anchor for microtubules (MT), which relates to peroxisome motility. For docking, the conserved N-terminal domain of PEX14 (PEX14-NTD) binds amphipathic alpha-helical ligands, typically comprising one or two aromatic residues, of which human PEX5 possesses eight. Here, we show that the PEX14-NTD also binds to microtubular filaments in vitro with a dissociation constant in nanomolar range. PEX14 interacts with two motifs in the C-terminal region of human ß-tubulin. At least one of the binding motifs is in spatial proximity to the binding site of microtubules (MT) for kinesin. Both PEX14 and kinesin can bind to MT simultaneously. Notably, binding of PEX14 to tubulin can be prevented by its association with PEX5. The data suggest that PEX5 competes peroxisome anchoring to MT by occupying the ß-tubulin-binding site of PEX14. The competitive correlation of matrix protein import and motility may facilitate the homogeneous dispersion of peroxisomes in mammalian cells.     

A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.     

A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported to the organelle by shuttling receptors. Matrix proteins containing a type 1 signal are carried to the peroxisome by PEX5, whereas those harboring a type 2 signal are transported by a PEX5-PEX7 complex. The pathway followed by PEX5 during the protein transport cycle has been characterized in detail. In contrast, not much is known regarding PEX7. In this work, we show that PEX7 is targeted to the peroxisome in a PEX5- and cargo-dependent manner, where it becomes resistant to exogenously added proteases. Entry of PEX7 and its cargo into the peroxisome occurs upstream of the first cytosolic ATP-dependent step of the PEX5-mediated import pathway, i.e., before monoubiquitination of PEX5. PEX7 passing through the peroxisome becomes partially, if not completely, exposed to the peroxisome matrix milieu, suggesting that cargo release occurs at the trans side of the peroxisomal membrane. Finally, we found that export of peroxisomal PEX7 back into the cytosol requires export of PEX5 but, strikingly, the two export events are not strictly coupled, indicating that the two proteins leave the peroxisome separately.     

The Arabidopsis pxa1 Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein Required for Peroxisomal Fatty Acid β-Oxidation

Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid beta-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid beta-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels beta-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for beta-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.     

MKP-1 Is Necessary for T Cell Activation and Function

MAPKs are evolutionarily conserved immune regulators. MAPK phosphatases (MKPs) that negatively regulate MAPK activities have recently emerged as critical players in both innate and adaptive immune responses. MKP-1, also known as DUSP1, was previously shown to negatively regulate innate immunity by inhibiting pro-inflammatory cytokine production. Here, we found that MKP-1 is necessary in T cell activation and function. MKP-1 deficiency in T cells impaired the activation, proliferation, and function of T cells in vitro, associated with enhanced activation of JNK and reduced NFATc1 translocation into the nucleus. Consistently, MKP-1^−/− mice were defective in anti-influenza immunity in vivo and resistant to experimental autoimmune encephalomyelitis. Our results thus demonstrate that MKP-1 is a critical positive regulator of T cell activation and function and may be targeted in treatment of autoimmune diseases.     

Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.     

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57–71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner.     

Copper Import into the Mitochondrial Matrix in Saccharomyces cerevisiae Is Mediated by Pic2, a Mitochondrial Carrier Family Protein

Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.     

Mutation of the Arabidopsis LON2 peroxisomal protease enhances pexophagy

Peroxisomes are critical organelles housing various, often oxidative, reactions. Pexophagy, the process by which peroxisomes are selectively targeted for destruction via autophagy, is characterized in yeast and mammals but had not been reported in plants. In this article, we describe how the peroxisome-related aberrations of a mutant defective in the LON2 peroxisomal protease are suppressed when autophagy is prevented by mutating any of several key autophagy-related (ATG) genes. Our results reveal that plant peroxisomes can be degraded by selective autophagy and suggest that pexophagy is accelerated when the LON2 protease is disabled.     

The Molecular Basis for Distinct Pathways for Protein Import into Arabidopsis Chloroplasts

The translocons at the outer envelope membrane of chloroplasts (TOCs) initiate the import of thousands of nucleus-encoded proteins into the organelle. The identification of structurally and functionally distinct TOC complexes has led to the hypothesis that the translocons constitute different import pathways that are required to coordinate the import of sets of proteins whose expression varies in response to organelle biogenesis and physiological adaptation. To test this hypothesis, we examined the molecular basis for distinct TOC pathways by analyzing the functional diversification among the Toc159 family of TOC receptors. We demonstrate that the N-terminal A-domains of the Toc159 receptors regulate their selectivity for preprotein binding. Furthermore, the in vivo function of the two major Toc159 family members (atToc159 and atToc132) can be largely switched by swapping their A-domains in transgenic Arabidopsis thaliana. On the basis of these results, we propose that the A-domains of the Toc159 receptors are major determinants of distinct pathways for protein import into chloroplasts.     

Prion Protein Is Necessary for Latent Learning and Long-Term Memory Retention

1. The cellular prion protein, designated PrP^c, is a key molecule in the prion diseases but its physiological function remains unknown. To elucidate whether PrP^c plays some role in the central nervous system, we established a line of mice in which the PrP gene had been disrupted and subsequently conducted long-term observations.2. Performance in latent learning and passive avoidance was evaluated using water-finding and step-through tests, respectively.3. PrP ^–/– mice showed impaired performance in the water-finding test, indicating a disturbance in latent learning, at 23 weeks of age. In the step-through test, although the PrP ^–/– mice showed normal learning ability and short-term memory retention, they evidenced a significant disturbance in long-term memory retention.4. These results indicate that PrP^c is needed for certain types of learning and memory and that the loss of function of this protein may contribute to the pathogenesis of prion diseases.