Chaperone and protease functions of LON protease 2 modulate the peroxisomal transition and degradation with autophagy.

Balancing repair and degradation is essential for maintaining organellar and cellular homeostasis. Peroxisomes are ubiquitous organelles in eukaryotic cells that play pivotal roles in cell survival. However, the quality control mechanism used to maintain peroxisomes is unclear. Here, we demonstrate that LON protease 2 (LON2), which is encoded by ABERRANT PEROXISOME MORPHOLOGY 10 (APEM10), is responsible for the functional transition of peroxisomes with autophagy. The Arabidopsis apem10 mutant displayed accelerated peroxisome degradation and a dramatically reduced number of peroxisomes. LON2 deficiency caused enhanced peroxisome degradation by autophagy, and peroxisomal proteins accumulated in the cytosol due to a decrease in the number of peroxisomes. We also show the proteolytic consequence of LON2 for the degradation of peroxisomal proteins, and we demonstrated that unnecessary proteins are eliminated by LON2- and autophagy-dependent degradation pathways during the functional transition of peroxisomes. LON2 plays dual roles as an ATP-dependent protease and a chaperone. We show that the chaperone domain of LON2 is essential for the suppression of autophagy, whereas its peptidase domain interferes with this chaperone function, indicating that intramolecular modulation between the proteolysis and chaperone functions of LON2 regulates degradation of peroxisomes by autophagy.

A defect of peroxisomal membrane protein 38 causes enlargement of peroxisomes.

Peroxisome proliferation occurs through enlargement, elongation and division of pre-existing peroxisomes. In the Arabidopsis apem mutant, apem3, peroxisomes are dramatically enlarged and reduced in number, revealing a defect in peroxisome proliferation. The APEM3 gene was found to encode peroxisomal membrane protein 38 (PMP38). To examine the relative role of PMP38 during proliferation, a double mutant was constructed consisting of apem3 and the peroxisome division mutant, apem1, in which a defect in dynamin-related protein 3A (DRP3A) results in elongation of peroxisomes. In the double mutant, almost all peroxisomes were predominantly enlarged but not elongated. DRP3A is still able to localize at the peroxisomal membrane on enlarged peroxisomes in the apem3 mutants. PMP38 is revealed to be capable of interacting with itself, but not with DRP3A. These results indicate that PMP38 has a role at a different step that requires APEM1/DRP3A. PMP38 is expressed in various tissues throughout the plant, indicating that PMP38 may participate in multiple unidentified functions in these tissues. PMP38 belongs to a mitochondrial carrier family (MCF) protein. However, unlike Arabidopsis nucleotide carrier protein 1 (AtPNC1) and AtPNC2, two other peroxisome-resident MCF proteins that function as adenine nucleotide transporters, PMP38 has no ATP or ADP transport activity. In addition, unlike AtPNC1 and AtPNC2 knock-down plants, apem3 mutants do not exhibit any gross morphological abnormalities. These results demonstrate that APEM3/PMP38 plays a role distinct from that of AtPNC1 and AtPNC2. We discuss possible mechanism of enlargement of peroxisomes in the apem3 mutants.

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes.

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H2O2 and ß-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein-protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.

Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation.

We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

A vacuolar iron transporter in tulip, TgVit1, is responsible for blue coloration in petal cells through iron accumulation.

Blue color in flowers is due mainly to anthocyanins, and a considerable part of blue coloration can be attributed to metal-complexed anthocyanins. However, the mechanism of metal ion transport into vacuoles and subsequent flower color development has yet to be fully explored. Previously, we studied the mechanism of blue color development specifically at the bottom of the inner perianth in purple tulip petals of Tulipa gesneriana cv. Murasakizuisho. We found that differences in iron content were associated with the development of blue- and purple-colored cells. Here, we identify a vacuolar iron transporter in T. gesneriana (TgVit1), and characterize the localization and function of this transporter protein in tulip petals. The amino acid sequence of TgVit1 is 85% similar that of the Arabidopsis thaliana vacuolar iron transporter AtVIT1, and also showed similarity to the AtVIT1 homolog in yeast, Ca(2+)-sensitive cross-complementer 1 (CCC1). The gene TgVit1 was expressed exclusively in blue-colored epidermal cells, and protein levels increased with increasing mRNA expression and blue coloration. Transient expression experiments revealed that TgVit1 localizes to the vacuolar membrane, and is responsible for the development of the blue color in purple cells. Expression of TgVit1 in yeast rescued the growth defect of ccc1 mutant cells in the presence of high concentrations of FeSO(4). Our results indicate that TgVit1 plays an essential role in blue coloration as a vacuolar iron transporter in tulip petals. These results suggest a new role for involvement of a vacuolar iron transporter in blue flower color development.

Plant catalase is imported into peroxisomes by Pex5p but is distinct from typical PTS1 import.

We have previously demonstrated that the targeting signal of pumpkin catalase, Cat1, is an internal PTS1 (peroxisomal targeting signal 1)-like sequence, QKL, located at -13 to -11 from the C-terminus, which is different from the typical PTS1 SKL motif located in the C-terminus. Here we show that Cat1 import into peroxisome is dependent on the cytosolic PTS receptor, Pex5p, in Arabidopsis, similar to typical PTS1 import, and that other components for transport of peroxisomal matrix proteins such as Pex14p, Pex13p, Pex12p and Pex10p also contribute to the import of Cat1. Interestingly, however, we found that Cat1 interacts with the N-terminal domain of Pex5p, but not the C-terminal domain for interaction with the typical PTS1, revealing that Pex5p recognizes Cat1 in a manner distinct from typical PTS1.

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes.

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.

An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division.

Peroxisomes undergo dramatic changes in size, shape, number, and position within the cell, but the division process of peroxisomes has not been characterized. We screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants). In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division. We showed that APM1 encodes dynamin-related protein 3A (DRP3A), and that mutations in APM1/DRP3A also caused aberrant morphology of mitochondria. The transient expression analysis showed that DRP3A is associated with the cytosolic side of peroxisomes. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants. We also report that the growth of Arabidopsis, which requires the cooperation of various organelles, including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles.

Distribution and characterization of peroxisomes in Arabidopsis by visualization with GFP: dynamic morphology and actin-dependent movement.

Peroxisomes were visualized in living cells of various tissues in transgenic Arabidopsis by green fluorescent protein (GFP) through the addition of the peroxisomal targeting signal 1 (PTS1) or PTS2. The observation using confocal laser scanning microscopy revealed that the GFP fluorescence signals were detected as spherical spots in all cells of two kinds of transgenic plants. Immunoelectron microscopic analysis using antibodies against the peroxisomal marker protein, catalase, showed the presence of GFP in peroxisomes, confirming that GFP was correctly transported into peroxisomes by PTS1 or PTS2 pathways. It has been also revealed that peroxisomes are motile organelles whose movement might be caused by cytoplasmic flow. The movement of peroxisomes was more prominent in root cells than that in leaves, and divided into two categories: a relatively slow, random, vibrational movement and a rapid movement. Treatment with anti-actin and anti-tubulin drugs revealed that actin filaments involve in the rapid movement of peroxisomes. Moreover, abnormal large peroxisomes are present as clusters at the onset of germination, and these clusters disappear in a few days. Interestingly, tubular peroxisomes were also observed in the hypocotyl. These findings indicate that the shape, size, number and movement of peroxisomes in living cells are dynamic and changeable rather than uniform.