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 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.     

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.     

A GH3-like domain in reaper is required for mitochondrial localization and induction of IAP degradation

Reaper is a potent pro-apoptotic protein originally identified in a screen for Drosophila mutants defective in apoptotic induction. Multiple functions have been ascribed to this protein, including inhibition of IAPs (inhibitors of apoptosis); induction of IAP degradation; inhibition of protein translation; and when expressed in vertebrate cells, induction of mitochondrial cytochrome c release. Structure/function analysis of Reaper has identified an extreme N-terminal motif that appears to be sufficient for inhibition of IAP function. We report here that this domain, although required for IAP destabilization, is not sufficient. Moreover, we have identified a small region of Reaper, similar to the GH3 domain of Grim, that is required for localization of Reaper to mitochondria, induction of IAP degradation, and potent cell killing. Although a mutant Reaper protein lacking the GH3 domain was deficient in these properties, these defects could be fully rectified by appending either the C-terminal mitochondrial targeting sequence from Bcl-xL or a homologous region from the pro-apoptotic protein HID. Together, these data strongly suggest that IAP destabilization by Reaper in intact cells requires Reaper localization to mitochondria and that induction of IAP instability by Reaper is important for the potent induction of apoptosis in Drosophila cells.     

FUNDC1 is a novel mitochondrial-associated-membrane (MAM) protein required for hypoxia-induced mitochondrial fission and mitophagy

Mitochondria need to be fragmented prior to engulfment by phagophores, the precursors to autophagosomes. However, how these 2 processes are finely regulated and integrated is poorly understood. We have shown that the outer mitochondrial membrane protein FUNDC1 is a novel mitochondrial-associated membrane (MAM) protein, enriched at the MAM by interacting with the ER resident protein CANX (calnexin) under hypoxia. As mitophagy proceeds, it dissociates from CANX and preferably recruits DNM1L/DRP1 to drive mitochondrial fission in response to hypoxic stress. In addition, knocking down of FUNDC1, DNM1L or CANX in hypoxic cells increases the number of elongated mitochondria and also reduces the colocalization of autophagosome and mitochondria, thus preventing mitophagy. These findings identify FUNDC1 as a molecular hub integrating mitochondrial fission and mitophagy at the MAM in response to hypoxia.     

A fission yeast RCC1-related protein is required for the mitosis to interphase transition.

The isolation and characterization of the mutant dcdts (defect in chromatin decondensation) has led to the identification of two conserved proteins required for the re-establishment of the interphase state following the completion of mitosis. The gene that rescues the dcdts mutant encodes a protein similar to the human chromatin binding protein, RCC1. A suppressor of dcdts encodes a protein nearly identical to the human GTP-binding protein, RAN, encoded by the TC4 gene. These results indicate that completion of mitosis is regulated at least in part by a GTPase molecular switch. The gene and suppressor of dcdts are identical to the previously described Schizosaccharomyces pombe genes pim1 (premature initiation of mitosis) and spi1 (suppressor of pim), but the dcdts mutant does not enter mitosis prematurely, a phenotype that has been reported for the pim1-46ts mutant. Based on our studies we propose that the pim1 gene product is required for regulating chromatin condensation with a primary role at the end of mitosis and pleiotropic effects on other aspects of cell behavior.     

ABORTED GAMETOPHYTE 1 is required for gametogenesis in Arabidopsis

In flowering plants, the male and female gametogenesis is a crucial step of sexual reproduction. Although many genes have been identi fied as being involved in the gametogenesis process, the genetic mechanisms underlying gametogenesis remains poorly understood. We reported here characterization of the gene, ABORTED GAMETOPHYTE 1(AOG1) that is newly identi fied as essential for gametogenesis in Arabidopsis thaliana. AOG1 is expressed predominantly in reproductive tissues including the developing pollen grains and ovules. The AOG1 protein shares no signi ficant amino acid sequence similarity with other documented proteins and is located mainly in nuclei of the cells. Mutation in AOG1 caused degeneration of pollen at the uninucleate microspore stage and severe defect in embryo sacs, leading to a signi ficant reduction in male and female fertility.Furthermore, the molecular analyses showed that the aog1 mutant signi ficantly affected the expression of several genes, which are required for gametogenesis. Our results suggest that AOG1 plays important roles in gameto genesis at the stage prior to pollen mitosis I(PMI)in Arabidopsis, possibly through collaboration with other genes.     

MOL1 is required for cambium homeostasis in Arabidopsis

Plants maintain pools of pluripotent stem cells which allow them to constantly produce new tissues and organs. Stem cell homeostasis in shoot and root tips depends on negative regulation by ligand–receptor pairs of the CLE peptide and leucine‐rich repeat receptor‐like kinase (LRR‐RLK) families. However, regulation of the cambium, the stem cell niche required for lateral growth of shoots and roots, is poorly characterized. Here we show that the LRR‐RLK MOL1 is necessary for cambium homeostasis in Arabidopsis thaliana. By employing promoter reporter lines, we reveal that MOL1 is active in a domain that is distinct from the domain of the positively acting CLE41/PXY signaling module. In particular, we show that MOL1 acts in an opposing manner to the CLE41/PXY module and that changing the domain or level of MOL1 expression both result in disturbed cambium organization. Underlining discrete roles of MOL1 and PXY, both LRR‐RLKs are not able to replace each other when their expression domains are interchanged. Furthermore, MOL1 but not PXY is able to rescue CLV1 deficiency in the shoot apical meristem. By identifying genes mis‐expressed in mol1 mutants, we demonstrate that MOL1 represses genes associated with stress‐related ethylene and jasmonic acid hormone signaling pathways which have known roles in coordinating lateral growth of the Arabidopsis stem. Our findings provide evidence that common regulatory mechanisms in different plant stem cell niches are adapted to specific niche anatomies and emphasize the importance of a complex spatial organization of intercellular signaling cascades for a strictly bidirectional tissue production.     

Arabidopsis PLC1 is required for secondary responses to abscisic acid signals

The role of inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) in transducing the abscisic acid (ABA) signal during seed germination and in the stress responses of mature plants is poorly understood. We have considered the contributions of the phospholipase C1 (encoded by AtPLC1) and an Ins(1,4,5)P3 5-phosphatase (encoded by AtIP5PII) to ABA signaling by using a modified version of the glucocorticoid-inducible system to regulate transgene expression. In the presence of the dexamethasone (Dex) inducer, transgenic lines expressing the AtPLC1 antisense and AtIP5PII sense transgenes showed no inhibition of germination and growth by ABA, whereas in the absence of the inducer they were sensitive. In the presence of Dex, these lines accumulated lower Ins(1,4,5)P3 levels upon ABA treatment compared with that of the control transgenic lines. RNA gel blot analysis revealed a decrease in the induction of the ABA-responsive genes RD29a, KIN2, and RD22 but not COR47 in the Dex-induced transgenic plants. In transgenic lines expressing the inducible AtPLC1 sense transgene, an increase in AtPLC1 expression was not sufficient to activate the expression of ABA-responsive genes in vegetative tissues. In vitro experiments demonstrated the induced PLC1 expression when extracts were assayed in the presence of calcium, but no increase in Ins(1,4,5)P3 levels in vivo was detected, suggesting that the PLC1 enzyme was latent. Our results indicate that although an increase in PLC1 activity and increased Ins(1,4,5)P3 levels are necessary for maximal gene induction by ABA, overexpression of AtPLC1 itself is not sufficient to trigger the expression of ABA-responsive genes. We propose that AtPLC1 plays a role in secondary ABA responses.     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast

Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.     

Gag3p, an outer membrane protein required for fission of mitochondrial tubules

Mitochondrial morphology and function depend on MGM1, a Saccharomyces cerevisiae gene encoding a dynamin-like protein of the mitochondrial outer membrane. Here, we show that mitochondrial fragmentation and mitochondrial genome loss caused by lesions in MGM1 are suppressed by three novel mutations, gag1, gag2, and gag3 (for glycerol-adapted growth). Cells with any of the gag mutations displayed aberrant mitochondrial morphology characterized by elongated, unbranched tubes and highly fenestrated structures. Additionally, each of the gag mutations prevented mitochondrial fragmentation caused by loss of the mitochondrial fusion factor, Fzo1p, or by treatment of cells with sodium azide. The gag1 mutation mapped to DNM1 that encodes a dynamin-related protein required for mitochondrial fission. GAG3 encodes a novel WD40-repeat protein previously found to interact with Dnm1p in a two-hybrid assay. Gag3p was localized to mitochondria where it was found to associate as a peripheral protein on the cytosolic face of the outer membrane. This association requires neither the DNM1 nor GAG2 gene products. However, the localization of Dnm1p to the mitochondrial outer membrane is substantially reduced by the gag2 mutation, but unaffected by loss of Gag3p. These results indicate that Gag3p plays a distinct role on the mitochondrial surface to mediate the fission of mitochondrial tubules.     

A novel protein kinase gene ssp1+ is required for alteration of growth polarity and actin localization in fission yeast.

Temperature-sensitive suppressor mutants were isolated from two fission yeast mutants defective in cell shape control: ppe1, encoding a type 2A-like protein phosphatase, and sts5, one of 11 staurosporine-supersensitive mutants. Complementation tests showed that suppression was due to two chromosomal loci, ssp1 and ssp2. Cells of the ssp1 mutant grown at the restrictive temperature arrested uniformly with an elongated cell body and a 2C content of DNA. Interestingly, these mutant cells grew only in a monopolar manner. At a specific point in the G2 phase of the cell cycle, wild-type cells exhibit a drastic alteration in growth polarity, from mono- to bipolar. This change coincides with the distribution of cortical actin from one end of the cell to both ends. In the ssp1 mutant cells, cortical actin was localized only at one end, suggesting that the mutant fails to change growth polarity. Nucleotide sequence determination showed that ssp1+ encodes a novel protein kinase. Ectopic overexpression of ssp1+ resulted in an altered cell morphology and cortical actin was randomly dispersed within the cells. Immunocytological analysis revealed that the protein was primarily localized in the cytoplasm and that half of the protein existed in an insoluble fraction. These results show that the dynamics of actin-based growth polarity during the cell cycle are regulated, at least in part, by a novel set of protein kinases and phosphatases.     

Arabidopsis MSI1 is required for epigenetic maintenance of reproductive development

WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism. Although MSI1-like proteins are well characterised biochemically, their function in the development of multicellular eukaryotes is not well understood. We constructed Arabidopsis plants in which the AtMSI1 protein level was altered. Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development. The primary shoot apical meristem was unable to develop organs after the transition to flowering. Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology, including a reduction in size of the petals and stamens and the development of carpel-like sepals. Ovule development was disrupted in all flowers, resulting in complete female sterility. Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2. In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes. Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly. Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.