PEX11alpha is required for peroxisome proliferation in response to 4-phenylbutyrate but is dispensable for peroxisome proliferator-activated receptor alpha-mediated peroxisome proliferation

The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.     

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.     

PEX11 promotes peroxisome division independently of peroxisome metabolism

The PEX11 peroxisomal membrane proteins are the only factors known to promote peroxisome division in multiple species. It has been proposed that PEX11 proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. Here we show that PEX11 proteins are unique in their ability to promote peroxisome division, and that PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. We also observed that mouse cells lacking PEX11beta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and that PEX11beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. Based on these and other observations, we propose that PEX11 proteins act directly in peroxisome division, and that their loss has indirect effects on peroxisome metabolism.     

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.     

Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A cooperate in cell cycle-associated replication of peroxisomes

Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle-associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in approximately 40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.     

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.     

The interaction between human PEX3 and PEX19 characterized by fluorescence resonance energy transfer (FRET) analysis

The process of peroxisome biogenesis involves several PEX genes that encode the machinery required to assemble the organelle. Among the corresponding peroxins the interaction between PEX3 and PEX19 is essential for early peroxisome biogenesis. However, the intracellular site of this protein interaction is still unclear. To address this question by fluorescence resonance energy transfer (FRET) analysis, we engineered the enhanced yellow fluorescent protein (EYFP) to the C-terminus of PEX3 and the enhanced cyan fluorescent protein (ECFP) to the N-terminus of PEX19. Functionality of the fusion proteins was shown by transfection of human PEX3- and PEX19-deficient fibroblasts from Zellweger patients with tagged versions of PEX3 and PEX19. This led to reformation of import-competent peroxisomes in both cell lines previously lacking detectable peroxisomal membrane structures. The interaction of PEX3-EYFP with ECFP-PEX19 in a PEX3-deficient cell line during peroxisome biogenesis was visualized by FRET imaging. Although PEX19 was predominantly localized to the cytoplasma, the peroxisome was identified to be the main intracellular site of the PEX3-PEX19 interaction. Results were confirmed and quantified by donor fluorescence photobleaching experiments. PEX3 deletion proteins lacking the N-terminal peroxisomal targeting sequence (PEX3 34-373-EYFP) or the PEX19-binding domain located in the C-terminal half of the protein (PEX3 1-140-EYFP) did not show the characteristic peroxisomal localization of PEX3, but were mislocalized to the cytoplasm (PEX3 34-373-EYFP) or to the mitochondria (PEX3 1-140-EYFP) and did not interact with ECFP-PEX19. We suggest that FRET is a suitable tool to gain quantitative spatial information about the interaction of peroxins during the process of peroxisome biogenesis in single cells. These findings complement and extend data from conventional in vitro protein interaction assays and support the hypothesis of PEX3 being an anchor for PEX19 at the peroxisomal membrane.     

Arabidopsis RING Peroxins are E3 Ubiquitin Ligases that Interact with Two Homologous Ubiquitin Receptor Proteins

Peroxisomes are essential eukaryotic organelles that mediate various metabolic processes. Peroxisome import depends on a group of peroxisome biogenesis factors called peroxins, many of which are evolutionarily conserved. PEX2, PEX10, and PEX12 are three RING-finger-domain-containing integral membrane peroxins crucial for protein import. In yeast (Saccharomyces cerevisae), RING peroxins act as E3 ligases, facilitating the recycling of the peroxisome import receptor protein PEX5 through ubiquitination. In plants, RING peroxins are essential to plant vitality. To elucidate the mode of action of the plant RING peroxins, we employed in vitro assays to show that the Arabidopsis RING peroxins also have E3 ligase activities. We also identified a PEX2-interacting protein, DSK2b, which is a member of the ubiquitin receptor family known to function as shuttle factors ferrying polyubiquitinated substrates to the proteasome for degradation. DSK2b and its tandem duplicate DSK2a are localized in the cytosol and the nucleus, and both interact with the RING domain of PEX2 and PEX12. DSK2 artificial microRNA lines did not display obvious defects in plant growth or peroxisomal processes, indicating functional redundancies among Arabidopsis ubiquitin receptor proteins. Our results suggest that Arabidopsis RING peroxins can function as E3 ligases and act together with the ubiquitin receptor protein DSK2 in the peroxisomal membrane-associated protein degradation system.     

Defective peroxisome membrane synthesis due to mutations in human PEX3 causes Zellweger syndrome, complementation group G

Zellweger cerebro-hepato-renal syndrome is a severe congenital disorder associated with defective peroxisomal biogenesis. At least 23 PEX genes have been reported to be essential for peroxisome biogenesis in various species, indicating the complexity of peroxisomal assembly. Cells from patients with peroxisomal biogenesis disorders have previously been shown to segregate into >/=12 complementation groups. Two patients assigned to complementation group G who had not been linked previously to a specific gene defect were confirmed as displaying a cellular phenotype characterized by a lack of even residual peroxisomal membrane structures. Here we demonstrate that this complementation group is associated with mutations in the PEX3 gene, encoding an integral peroxisomal membrane protein. Homozygous PEX3 mutations, each leading to C-terminal truncation of PEX3, were identified in the two patients, who both suffered from a severe Zellweger syndrome phenotype. One of the mutations involved a single-nucleotide insertion in exon 7, whereas the other was a single-nucleotide substitution eight nucleotides from the normal splice site in the 3' acceptor site of intron 10. Expression of wild-type PEX3 in the mutant cell lines restored peroxisomal biogenesis, whereas transfection of mutated PEX3 cDNA did not. This confirmed that the causative gene had been identified. The observation of peroxisomal formation in the absence of morphologically recognizable peroxisomal membranes challenges the theory that peroxisomes arise exclusively by growth and division from preexisting peroxisomes and establishes PEX3 as a key factor in early human peroxisome synthesis.     

The Role of Conserved PEX3 Regions in PEX19-Binding and Peroxisome Biogenesis

The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.     

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.     

PEX3 is the causal gene responsible for peroxisome membrane assembly-defective Zellweger syndrome of complementation group G

Peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome (ZS) and neonatal adrenoleukodystrophy are autosomal recessive diseases caused by defects in peroxisome assembly, for which 13 genotypes have been identified. Expression of the human peroxin Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in fibroblasts from PBDG-02, a patient with complementation group G (CG-G) ZS. Patient PBDG-02 carried a homozygous, inactivating mutation-a 97-bp deletion of nucleotide residues at positions 942-1038-resulting in a 32-amino-acid truncation and in a frameshift inducing both a 3-amino-acid substitution and a termination codon. Genomic PCR analysis revealed mutation of T-->G at eight bases upstream of the splicing site at the boundary of intron 10 and exon 11 of PEX3 gene, giving rise to a deletion of all of exon 11. When assessed by expression in a pex3 mutant of Chinese hamster ovary cells and the patient's fibroblasts, PBDG-02-derived PEX3 cDNA was found to be defective in peroxisome-restoring activity. These results provide evidence that PEX3 is a novel, pathogenic gene responsible for CG-G PBDs.     

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.     

Reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p involves the nuclear envelope

The reassembly of peroxisomes in Hansenula polymorpha pex3 cells on reintroduction of Pex3p was examined. Using a Pex3-green fluorescent protein (Pex3-GFP) fusion protein, expressed under the control of an inducible promoter, it was observed that, initially on induction of Pex3-GFP synthesis, GFP fluorescence was localized to the endoplasmic reticulum and the nuclear envelope. Subsequently, a single organelle developed per cell that increased in size and multiplied by division. At these stages, GFP fluorescence was confined to peroxisomes. Fractionation experiments on homogenates of pex3 cells, in which the endoplasmic reticulum and nuclear envelope were marked with GFP, identified a small amount of GFP in peroxisomes present in the initial stage of peroxisome reassembly. Our data suggest a crucial role for the endoplasmic reticulum/nuclear envelope in peroxisome reintroduction on complementation of pex3 cells by the PEX3 gene.     

The Early-Acting Peroxin PEX19 Is Redundantly Encoded,Farnesylated, and Essential for Viability in Arabidopsis thaliana

Peroxisomes are single-membrane bound organelles that are essential for normal development in plants and animals. In mammals and yeast, the peroxin (PEX) proteins PEX3 and PEX19 facilitate the early steps of peroxisome membrane protein (PMP) insertion and pre-peroxisome budding from the endoplasmic reticulum. The PEX3 membrane protein acts as a docking site for PEX19, a cytosolic chaperone for PMPs that delivers PMPs to the endoplasmic reticulum or peroxisomal membrane. PEX19 is farnesylated in yeast and mammals, and we used immunoblotting with prenylation mutants to show that PEX19 also is fully farnesylated in wild-type Arabidopsis thaliana plants. We examined insertional alleles disrupting either of the two Arabidopsis PEX19 isoforms, PEX19A or PEX19B, and detected similar levels of PEX19 protein in the pex19a-1 mutant and wild type; however, PEX19 protein was nearly undetectable in the pex19b-1 mutant. Despite the reduction in PEX19 levels in pex19b-1, both pex19a-1 and pex19b-1 single mutants lacked notable peroxisomal β-oxidation defects and displayed normal levels and localization of peroxisomal matrix and membrane proteins. The pex19a-1 pex19b-1 double mutant was embryo lethal, indicating a redundantly encoded critical role for PEX19 during embryogenesis. Expressing YFP-tagged versions of either PEX19 isoform rescued this lethality, confirming that PEX19A and PEX19B act redundantly in Arabidopsis. We observed that pex19b-1 enhanced peroxisome-related defects of a subset of peroxin-defective mutants, supporting a role for PEX19 in peroxisome function. Together, our data indicate that Arabidopsis PEX19 promotes peroxisome function and is essential for viability.     

Proteomic Analysis Reveals That the Rab GTPase RabE1c Is Involved in the Degradation of the Peroxisomal Protein Receptor PEX7 (Peroxin 7)

The biogenesis of peroxisomes is mediated by peroxins (PEXs). PEX7 is a cytosolic receptor that imports peroxisomal targeting signal type 2 (PTS2)-containing proteins. Although PEX7 is important for protein transport, the mechanisms that mediate its function are unknown. In this study, we performed proteomic analysis to identify PEX7-binding proteins using transgenic Arabidopsis expressing green fluorescent protein (GFP)-tagged PEX7. Our analysis identified RabE1c, a small GTPase, as a PEX7 binding partner. In vivo analysis revealed that GTP-bound RabE1c binds to PEX7 and that a subset of RabE1c localizes to peroxisomes and interacts with PEX7 on the peroxisome membrane. Unlike endogenous PEX7, which is predominantly localized to the cytosol, GFP-PEX7 accumulates abnormally on the peroxisomal membrane and induces degradation of endogenous PEX7, concomitant with a reduction in import of PTS2-containing proteins and decreased peroxisomal β-oxidation activity. Thus, GFP-PEX7 on the peroxisomal membrane exerts a dominant negative effect. Mutation of RabE1c restored endogenous PEX7 protein expression and import of PTS2-containing proteins as well as peroxisomal β-oxidation activity. Treatment with proteasome inhibitors also restored endogenous PEX7 protein levels in GFP-PEX7-expressing seedlings. Based on these findings, we conclude that RabE1c binds PEX7 and facilitates PEX7 degradation in the presence of immobile GFP-PEX7 accumulated at the membrane.