The Arabidopsis Chloroplast Division Protein DYNAMIN-RELATED PROTEIN5B Also Mediates Peroxisome Division

Peroxisomes are highly dynamic organelles involved in various metabolic pathways. The division of peroxisomes is regulated by factors such as the PEROXIN11 (PEX11) proteins that promote peroxisome elongation and the dynamin-related proteins (DRPs) and FISSION1 (FIS1) proteins that function together to mediate organelle fission. In Arabidopsis thaliana, DRP3A/DRP3B and FIS1A (BIGYIN)/FIS1B are two pairs of homologous proteins known to function in both peroxisomal and mitochondrial division. Here, we report that DRP5B, a DRP distantly related to the DRP3s and originally identified as a chloroplast division protein, also contributes to peroxisome division. DRP5B localizes to both peroxisomes and chloroplasts. Mutations in the DRP5B gene lead to peroxisome division defects and compromised peroxisome functions. Using coimmunoprecipitation and bimolecular fluorescence complementation assays, we further demonstrate that DRP5B can interact or form a complex with itself and with DRP3A, DRP3B, FIS1A, and most of the Arabidopsis PEX11 isoforms. Our data suggest that, in contrast with DRP3A and DRP3B, whose orthologs exist across plant, fungal, and animal kingdoms, DRP5B is a plant/algal invention to facilitate the division of their organelles (i.e., chloroplasts and peroxisomes). In addition, our results support the notion that proteins involved in the early (elongation) and late (fission) stages of peroxisome division may act cooperatively.     

Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis

Peroxisomes are multi-functional organelles that differ in size and abundance depending on the species, cell type, developmental stage, and metabolic and environmental conditions. The PEROXIN11 protein family and the DYNAMIN-RELATED PROTEIN3A (DRP3A) protein have been shown previously to play key roles in peroxisome division in Arabidopsis. To establish a mechanistic model of peroxisome division in plants, we employed forward and reverse genetic approaches to identify more proteins involved in this process. In this study, we identified three new components of the Arabidopsis peroxisome division apparatus: DRP3B, a homolog of DRP3A, and FISSION1A and 1B (FIS1A and 1B), two homologs of the yeast and mammalian FIS1 proteins that mediate the fission of peroxisomes and mitochondria by tethering the DRP proteins to the membrane. DRP3B is partially targeted to peroxisomes and causes defects in peroxisome fission when the gene function is disrupted. drp3A drp3B double mutants display stronger deficiencies than each single mutant parent with respect to peroxisome abundance, seedling establishment and plant growth, suggesting partial functional redundancy between DRP3A and DRP3B. In addition, FIS1A and FIS1B are each dual-targeted to peroxisomes and mitochondria; their mutants show growth inhibition and contain peroxisomes and mitochondria with incomplete fission, enlarged size and reduced number. Our results demonstrate that both DRP3 and FIS1 protein families contribute to peroxisome fission in Arabidopsis, and support the view that DRP and FIS1 orthologs are common components of the peroxisomal and mitochondrial division machineries in diverse eukaryotic species.     

The PEROXIN11 protein family controls peroxisome proliferation in Arabidopsis

PEROXIN11 (PEX11) is a peroxisomal membrane protein in fungi and mammals and was proposed to play a major role in peroxisome proliferation. To begin understanding how peroxisomes proliferate in plants and how changes in peroxisome abundance affect plant development, we characterized the extended Arabidopsis thaliana PEX11 protein family, consisting of the three phylogenetically distinct subfamilies PEX11a, PEX11b, and PEX11c to PEX11e. All five Arabidopsis PEX11 proteins target to peroxisomes, as demonstrated for endogenous and cyan fluorescent protein fusion proteins by fluorescence microscopy and immunobiochemical analysis using highly purified leaf peroxisomes. PEX11a and PEX11c to PEX11e behave as integral proteins of the peroxisome membrane. Overexpression of At PEX11 genes in Arabidopsis induced peroxisome proliferation, whereas reduction in gene expression decreased peroxisome abundance. PEX11c and PEX11e, but not PEX11a, PEX11b, and PEX11d, complemented to significant degrees the growth phenotype of the Saccharomyces cerevisiae pex11 null mutant on oleic acid. Heterologous expression of PEX11e in the yeast mutant increased the number and reduced the size of the peroxisomes. We conclude that all five Arabidopsis PEX11 proteins promote peroxisome proliferation and that individual family members play specific roles in distinct peroxisomal subtypes and environmental conditions and possibly in different steps of peroxisome proliferation.     

The Arabidopsis tail-anchored protein PEROXISOMAL AND MITOCHONDRIAL DIVISION FACTOR1 is involved in the morphogenesis and proliferation of peroxisomes and mitochondria

Peroxisomes and mitochondria are multifunctional eukaryotic organelles that are not only interconnected metabolically but also share proteins in division. Two evolutionarily conserved division factors, dynamin-related protein (DRP) and its organelle anchor FISSION1 (FIS1), mediate the fission of both peroxisomes and mitochondria. Here, we identified and characterized a plant-specific protein shared by these two types of organelles. The Arabidopsis thaliana PEROXISOMAL and MITOCHONDRIAL DIVISION FACTOR1 (PMD1) is a coiled-coil protein tethered to the membranes of peroxisomes and mitochondria by its C terminus. Null mutants of PMD1 contain enlarged peroxisomes and elongated mitochondria, and plants overexpressing PMD1 have an increased number of these organelles that are smaller in size and often aggregated. PMD1 lacks physical interaction with the known division proteins DRP3 and FIS1; it is also not required for DRP3's organelle targeting. Affinity purifications pulled down PMD1's homolog, PMD2, which exclusively targets to mitochondria and plays a specific role in mitochondrial morphogenesis. PMD1 and PMD2 can form homo- and heterocomplexes. Organelle targeting signals reside in the C termini of these proteins. Our results suggest that PMD1 facilitates peroxisomal and mitochondrial proliferation in a FIS1/DRP3-independent manner and that the homologous proteins PMD1 and PMD2 perform nonredundant functions in organelle morphogenesis.     

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively. PEX26 responsible for peroxisome biogenesis disorders of complementation group 8 codes for C-tail-anchored type-II membrane peroxin Pex26p, the recruiter of Pex1p-Pex6p complexes to peroxisomes. Pex1p is targeted to peroxisomes in a manner dependent on ATP hydrolysis, while Pex6p targeting requires ATP but not its hydrolysis. Pex1p and Pex6p are most likely regulated in their peroxisomal localization onto Pex26p via conformational changes by ATPase cycle. Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome targeting signal type-1 and shuttles between the cytosol and peroxisomes. AAA peroxins are involved in the export from peroxisomes of Pex5p. Pex5p is ubiquitinated at the conserved cysteine11 in a form associated with peroxisomes. Pex5p with a mutation of the cysteine11 to alanine, termed Pex5p-C11A, abrogates peroxisomal import of proteins harboring peroxisome targeting signals 1 and 2 in wild-type cells. Pex5p-C11A is imported into peroxisomes but not exported, hence suggesting an essential role of the cysteine residue in the export of Pex5p.     

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.     

Plant Peroxisome Multiplication： Highly Regulatec and Still Enigmatic

Plant peroxisomes play a key role in numerous physiological processes and are able to adapt to environmental changes by altering their content, morphology, and abundance. Peroxisomes can multiply through elongation, constriction, and fission; this process requires the action of conserved, as well as species-specific proteins. Genetic and morphological analyses have been used with the model plant Arabidopsis thaliana to determine at the mechanistic level how plant peroxisomes increase their abundance. The five-member PEXll family promotes early steps of peroxisome multiplication with an unknown mechanism and some subfamily specificities. The dynamin-related protein （DRP）3 subfamily of dynaminrelated large guanosine triphosphatases mediates late steps of both peroxisomal and mitochondrial multiplication. New genetic and biochemical tools will be needed to identify additional, especially plant-specific, constituents of the peroxisome multiplication pathways.     

The conserved fission complex on peroxisomes and mitochondria

Peroxisomes are eukaryotic organelles highly versatile and dynamic in content and abundance. Plant peroxisomes mediate various metabolic pathways, a number of which are completed sequentially in peroxisomes and other subcellular organelles, including mitochondria and chloroplasts. To understand how peroxisomal dynamics contribute to changes in plant physiology and adaptation, the multiplication pathways of peroxisomes are being dissected. Research in Arabidopsis thaliana has identified several evolutionarily conserved families of proteins in peroxisome division. These include five PEROXIN11 proteins (PEX11a to -e) that induce peroxisome elongation and the fission machinery, which is composed of three dynamin-related proteins (DRP3A, -3B and -5B) and DRP''s membrane receptor, FISSION1 (FIS1A and -1B). While the function of PEX11 is restricted to peroxisomes, the fission factors are more promiscuous. DRP3 and FIS1 proteins are shared between peroxisomes and mitochondria, and DRP5B plays a dual role in the division of chloroplasts and peroxisomes. Analysis of the Arabidopsis genome suggests that higher plants may also contain functional homologs of the yeast Mdv1/Caf4 proteins, adaptor proteins that link DRPs to FIS1 on the membrane of both peroxisomes and mitochondria. Sharing a conserved fission machine between these metabolically linked subcellular compartments throughout evolution may have some biological significance.Key words: Arabidopsis, peroxisomal and mitochondrial division, dynamin-related protein (DRP), FISSION1 (FIS1), mitochondrial division 1 (Mdv1), CCR4p-associated factor 4 (Caf4)Peroxisomes are single membrane-delimited organelles involved in a variety of metabolic pathways essential to development.¹ Plant peroxisomes participate in processes such as lipid mobilization, photorespiration, detoxification, hormone biosynthesis and metabolism, and plant-pathogen interaction.^2,3 A number of these metabolic functions, such as photorespiration, fatty acid metabolism and jasmonic acid biosynthesis, are accomplished through the cooperative efforts of peroxisomes and other subcellular compartments, such as mitochondria and chloroplasts.^3–5 The function, morphology and abundance of peroxisomes can vary depending on the organism, cell type, developmental stage and prevailing environmental conditions in which the organism resides.^6,7 It is now believed that in addition to budding from the endoplasmic reticulum (ER), peroxisomes also multiply from pre-existing peroxisomes via division, going through steps including peroxisome elongation/tubulation, membrane constriction and fission.^7,8In the reference plant Arabidopsis thaliana, three evolutionarily conserved families of proteins have been identified as key components of the peroxisome division apparatus. Five integral membrane proteins, named PEX11a to -e, are mainly responsible for inducing the elongation and tubulation of peroxisomes in the early stage of peroxisome division.^9–11 DRP3A and DRP3B are members of a dynamin-related protein family that powers the fission of membranes and FIS1A and FIS1B are homologous proteins believed to anchor the DRP proteins to the membrane.^12–19 Similar to their counterparts in yeasts and mammals, DRP3 and FIS1 are shared by the fission machineries of peroxisomes and mitochondria.^12–19 We recently reported the unexpected finding that DRP5B, a plant/algal-specific DRP distantly related to the DRP3 proteins and originally discovered for its function in chloroplast division, is also involved in the division of peroxisomes. Using co-immunoprecipitation (co-IP) and bimolecular fluorescence complementation (BiFC) assays, we further demonstrated that DRP5B and the two DRP3 proteins can homo- and hetero-dimerize and each DRP can form a complex with FIS1A and/or FIS1B and most of the Arabidopsis PEX11 isoforms.²⁰ These results together demonstrate that, despite their distinct evolutionary origins, structures and functions, peroxisomes, mitochondria and chloroplasts use some of the same factors for fission. These data also revealed that, like in yeasts and mammals, the FIS1-DRP complex exits on peroxisomes and mitochondria in plants.DRP5B, a DRP unique in the plant and photosynthetic algae lineages, seems to be the sole component shared by the division of chloroplasts and peroxisomes.²⁰ However, both FIS1 and DRP are found to be required for the division of peroxisomes and mitochondria throughout eukaryotic evolution,^21,22 prompting the question: to what extent is the FIS1-DRP complex conserved among diverse species? In the yeast Saccharomyces cerevisiae, this fission complex also contains an adaptor encoded by two homologous WD40 proteins, Mdv1 and Caf4, which are partially redundant in function with Mdv1 playing the major role. Mdv1 and Caf4 share an N-terminal extension (NTE) domain with two α-helices, a middle coiled-coil domain (CC) and C-terminal WD40 repeat. Both proteins use the NTE to interact with the tetratricopeptide repeat (TPR) domain-containing N-terminus of Fis1, the CC domain to dimerize and the C-terminal WD40 repeat to interact with and recruit the DRP protein, Dnm1.^23,24 The Hansenula polymorpha Mdv1 (Hp Mdv1) also has a dual function in the division of peroxisomes and mitochondria.²⁵ In addition, a Mdv1/Caf4 homolog, Mda1, was identified from the primitive red algae Cyanidioschyzon merolae and found to be involved at least in mitochondrial fission.²⁶ However, higher eukaryotes do not seem to have obvious homologs of Mdv1/Caf4. For example, mammals contain Fis1 and Drp (called DLP1 or Drp1) but no apparent homologs to Mdv1 and Caf4. Instead, a metazoan-specific tail-anchored protein, Mitochondrial Fission Factor (Mff), was recently found to regulate the fission of mitochondria and peroxisomes in a similar manner to Fis1. Mff is essential in recruiting Drp1, at least in mitochondrial division, yet it functions in a Fis1-independent pathway.^27,28To determine whether plants contain structural or functional homologs of Mdv1 and Caf4, we performed blast searches of the Arabidopsis genome, which resulted in the retrieval of ∼300 WD40 proteins. However, just like the search results from mammals, none of these proteins show significant sequence similarity with Mdv1 and Caf4 beyond the WD40 repeats. To identify proteins with similar domain structures with Mdv1/Caf4, we further analyzed these WD40 proteins, using the online Simple Modular Architecture Research Tool (smart.embl-heidelberg.de/). After eliminating proteins apparently inappropriate to be part of this complex, such as kinases and proteins with drastically distinct domain organizations despite of having both WD40 repeats and CC domains, we were able to narrow down to eight proteins. These proteins, which are encoded by At1g04510, At2g32950, At2g33340, At3g18860, At4g05410, At4g21130, At5g50230 and At5g67320, respectively, each contain a central CC domain in addition to the WD40 repeat region and are ranging from 450 to 900 amino acids in length (Fig. 1A). Subcellular localization studies will need to be performed to determine whether some of these proteins are associated with peroxisomes and mitochondria. If such a WD40 protein is proven to be part of the FIS1-DRP complex in Arabidopsis, it will be important to determine whether it simply acts as an adaptor or it also plays other roles, such as to promote and maintain the active structure and conformation of DRP3A/3B at the division site (Fig. 1B). Consistent with the latter scenario, it was found that Sc Mdv1 accumulates at the division sites after Dnm1 assembles and that the mammalian Fis1 and Drp1 proteins physically interact.^29,30 Peroxisomes and mitochondria are functionally linked in a number of metabolic pathways. For example, in plants, they act cooperatively in important processes such as fatty acid metabolism and photorespiration.³ An interesting question to address in the future is whether sharing such a conserved fission machine between peroxisomes and mitochondria throughout evolution has critical biological consequences.Open in a separate windowFigure 1Domain structure of Mdv1/Caf4 and their homologs or putative homologs. (A) Domain structure of Sc Mdv1 and Sc Caf4 from S. cerevisiae, their homologs from H. polymorpha and C. merolae, and the eight Arabidopsis proteins with similar domain organization. Grey boxes indicate the CC domain and black boxes are Wd40 repeats. (B) The putative FIS1-WD40-DRP complex in Arabidopsis. CC, coiled-coil; NTE, N-terminal extension; TPR, tetratricopeptide repeat; TMD, transmembrane domain.     

The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.     

Peroxisome reintroduction in Hansenula polymorpha requires Pex25 and Rho1

We identified two proteins, Pex25 and Rho1, which are involved in reintroduction of peroxisomes in peroxisome-deficient yeast cells. These are, together with Pex3, the first proteins identified as essential for this process. Of the three members of the Hansenula polymorpha Pex11 protein family-Pex11, Pex25, and Pex11C-only Pex25 was required for reintroduction of peroxisomes into a peroxisome-deficient mutant strain. In peroxisome-deficient pex3 cells, Pex25 localized to structures adjacent to the ER, whereas in wild-type cells it localized to peroxisomes. Pex25 cells were not themselves peroxisome deficient but instead contained a slightly increased number of peroxisomes. Interestingly, pex11 pex25 double deletion cells, in which both peroxisome fission (due to the deletion of PEX11) and reintroduction (due to deletion of PEX25) was blocked, did display a peroxisome-deficient phenotype. Peroxisomes reappeared in pex11 pex25 cells upon synthesis of Pex25, but not of Pex11. Reintroduction in the presence of Pex25 required the function of the GTPase Rho1. These data therefore provide new and detailed insight into factors important for de novo peroxisome formation in yeast.     

Inhibitors of COPI and COPII do not block PEX3-mediated peroxisome synthesis

In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.     

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.     

The Arabidopsis peroxisome division mutant pdd2 is defective in the DYNAMIN-RELATED PROTEIN3A (DRP3A) gene

In plants, the division of peroxisomes is mediated by several classes of proteins, including PEROXIN11 (PEX11), FISSION1 (FIS1) and DYNAMIN-RELATED PROTEIN3 (DRP3). DRP3A and DRP3B are two homologous dynamin-related proteins playing overlapping roles in the division of both peroxisomes and mitochondria, with DRP3A performing a stronger function than DRP3B in peroxisomal fission. Here, we report the identification and characterization of the peroxisome division defective 2 (pdd2) mutant, which was later proven to be another drp3A allele. The pdd2 mutant generates a truncated DRP3A protein and exhibits pale green and retarded growth phenotypes. Intriguingly, this mutant displays much stronger peroxisome division deficiency in root cells than in leaf mesophyll cells. Our data suggest that the partial GTPase effector domain retained in pdd2 may have contributed to the distinct mutant phenotype of this mutant.Key words: peroxisome division, dynamin-related protein, arabidopsisIn eukaryotic cells, peroxisomes are surrounded by single membranes and house a variety of oxidative metabolic pathways such as lipid metabolism, detoxification and plant photorespiration.^1,2 To accomplish multiple tasks, the morphology, abundance and positioning of peroxisomes need to be highly regulated. Three families of proteins, whose homologs are present across different kingdoms, have been shown to be involved in peroxisome division in Arabidopsis. The PEX11 protein family is composed of five integral membrane proteins with primary roles in peroxisome elongation/tubulation, the initial step in peroxisome division.^3–5 Although the exact function of PEX11s has not been demonstrated, these proteins are believed to participate in peroxisome membrane modification.^6,7 The FIS1 family consists of two isoforms, which are C-terminal tail-anchored membrane proteins with rate limiting functions at the fission step.^8,9 DRP3A and DRP3B belong to a superfamily of dynamin-related proteins, which are large and self-assembling GTPases involved in the fission and fusion of membranes by acting as mechanochemical enzymes or signaling GTPases.¹⁰ The function of PEX11 seems to be exclusive to peroxisomes, whereas DRP3 and FIS1 are shared by the division machineries of both peroxisomes and mitochondria in Arabidopsis.^8,9,11–16 FIS1 proteins are believed to tether DRP proteins to the peroxisomal membrane,^17,18 but direct evidence has not been obtained from plants. DRP3A and DRP3B share 77% sequence identity at the protein level and are functionally redundant in regulating mitochondrial division; however, DRP3A''s role on the peroxisome seems stronger and cannot be substituted by DRP3B in peroxisome division.^8,13,15In a continuous effort to identify components of the plant peroxisome division apparatus from Arabidopsis, we performed genetic screens in a peroxisomal marker background expressing the YFP (yellow fluorescent protein)-PTS1 (peroxisome targeting signal 1, containing Ser-Lys-Leu) fusion protein. Mutants with defects in the morphology and abundance of fluorescently labeled peroxisomes are characterized. Following our analysis of the pdd1 mutant, which turned out to be a strong allele of DRP3A,⁸ we characterized the pdd2 mutant.In root cells of the pdd2 mutant, extremely elongated peroxisomes and a beads-on-a-string peroxisomal phenotype are frequently observed (Fig. 1A and B). These peroxisome phenotypes resemble those of pdd1 and other strong drp3A alleles previously reported.^8,15 However, the peroxisome phenotype seems to be less dramatic in leaf mesophyll cells. For instance, in addition to the decreased number of total peroxisomes, peroxisomes in leaf cells are only slightly elongated or exhibit a beads-on-a-string phenotype (Fig. 1C and D). Previously, we reported the phenotypes of three strong drp3A alleles, all of which contain a large number of peroxules, long and thin membrane extensions from the peroxisome,⁸ yet such peroxisomal structures are not observed in pdd2. On the other hand, pdd2 has a more severe growth phenotype than most drp3A alleles, as it is slow in growth and has pale green leaves (Fig. 1E). Genetic analysis showed that pdd2 segregates as a single recessive mutation (data not shown).Open in a separate windowFigure 1Phenotypic analyses of pdd2 and identification of the PDD2 gene. (A–D) Confocal micrographs of root and mesophyll cells in 3-week-old wild type and pdd2 mutant plants. Green signals show peroxisomes; red signals show chloroplasts. Scale bars = 20 µm. (E) Growth phenotype of 3-week-old mutants. (F) Map-based cloning of the PDD2 gene. Genetic distance from PDD2 is shown under each molecular marker. Positions for mutations in previously analyzed drp3A alleles and pdd2 are indicated in the gene schematic. drp3A-1 and drp3A-2 are T-DNA insertion mutants, whereas pdd1 is an EMS mutant containing a premature stop codon in exon 6. (G) A schematic of the DRP3A (PDD2) protein with functional domains indicated. The pdd2 allele encodes a truncated protein lacking part of the GED domain.The unique combination of peroxisomal and growth phenotypes of pdd2 prompted us to use map-based cloning to identify the PDD2 gene, with the hope to discover novel proteins in the peroxisome division machinery. A population of approximately 6,000 F₂ plants (pdd2 × Ler) was generated. After screening 755 F₂ mutants, the pdd2 mutation was mapped to the region between markers T10C21 and F4B14 on the long arm of chromosome 4 (Fig. 1F). Since this region contains DRP3A, we sequenced the entire DRP3A gene in pdd2 and identified a G→A transition at the junction of the 18^th exon and intron (Fig. 1F). Further analysis revealed that the point mutation at this junction caused mis-splicing of intron 18, introducing a stop codon in the GTPase effector domain GED near the C terminus (Fig. 1G).DRPs share with the classic dynamins an N-terminal GTPase domain, a middle domain (MD), and a regulatory motif named the GTPase effector domain (GED) (Fig. 1G). To date, a total of 26 drp3A mutant alleles carrying missense or nonsense mutations along the length of the DRP3A gene have been isolated.^8,15 The combined peroxisomal and growth phenotype of pdd2 and the nature of the mutation in this allele are unique among all the drp3A alleles, indicating that the partial GED domain retained in pdd2 may have created some novel function for this protein. Further analysis of the truncated protein may be necessary to test this prediction.     

Fis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis

Dynamin-like protein 1 (DLP1) and Pex11pbeta function in morphogenesis of peroxisomes. In the present work, we investigated whether Fis1 is involved in fission of peroxisomes. Endogenous Fis1 was morphologically detected in peroxisomes as well as mitochondria in wild-type CHO-K1 and DLP1-defective ZP121 cells. Subcellular fractionation studies also revealed the presence of Fis1 in peroxisomes. Peroxisomal Fis1 showed the same topology, i.e., C-tail anchored membrane protein, as the mitochondrial one. Furthermore, ectopic expression of FIS1 induced peroxisome proliferation in CHO-K1 cells, while the interference of FIS1 RNA resulted in tubulation of peroxisomes, hence reducing the number of peroxisomes. Fis1 interacted with Pex11pbeta, by direct binding apparently involving the C-terminal region of Pex11pbeta in the interaction. Pex11pbeta also interacted with each other, whereas the binding of Pex11pbeta to DLP1 was not detectable. Moreover, ternary complexes comprising Fis1, Pex11pbeta, and DLP1 were detected by chemical cross-linking. We also showed that the highly conserved N-terminal domain of Pex11pbeta was required for the homo-oligomerization of Pex11pbeta and indispensable for the peroxisome-proliferating activity. Taken together, these findings indicate that Fis1 plays important roles in peroxisome division and maintenance of peroxisome morphology in mammalian cells, possibly in a concerted manner with Pex11pbeta and DLP1.     

Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis “master-regulator” PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.     

Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.     

Peroxisome proliferation in Hansenula polymorpha requires Dnm1p which mediates fission but not de novo formation

We show that the dynamin-like proteins Dnm1p and Vps1p are not required for re-introduction of peroxisomes in Hansenula polymorpha pex3 cells upon complementation with PEX3-GFP. Instead, Dnm1p, but not Vps1p, plays a crucial role in organelle proliferation via fission. In H. polymorpha DNM1 deletion cells (dnm1) a single peroxisome is present that forms long extensions, which protrude into developing buds and divide during cytokinesis. Budding pex11.dnm1 double deletion cells lack these peroxisomal extensions, suggesting that the peroxisomal membrane protein Pex11p is required for their formation. Life cell imaging revealed that fluorescent Dnm1p-GFP spots fluctuate between peroxisomes and mitochondria. On the other hand Pex11p is present over the entire organelle surface, but concentrates during fission at the basis of the organelle extension in dnm1 cells.Our data indicate that peroxisome fission is the major pathway for peroxisome multiplication in H. polymorpha.