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1.  Functional Investigation of the Plant-Specific Long Coiled-Coil Proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL) in Arabidopsis thaliana 
PLoS ONE  2013;8(2):e57283.
We have identified and characterized two Arabidopsis long coiled-coil proteins PAMP-INDUCED COILED-COIL (PICC) and PICC-LIKE (PICL). PICC (147 kDa) and PICL (87 kDa) are paralogs that consist predominantly of a long coiled-coil domain (expanded in PICC), with a predicted transmembrane domain at the immediate C-terminus. Orthologs of PICC and PICL were found exclusively in vascular plants. PICC and PICL GFP fusion proteins are anchored to the cytoplasmic surface of the endoplasmic reticulum (ER) membrane by a C-terminal transmembrane domain and a short tail domain, via a tail-anchoring mechanism. T-DNA-insertion mutants of PICC and PICL as well as the double mutant show an increased sensitivity to the plant abiotic stress hormone abscisic acid (ABA) in a post-germination growth response. PICC, but not PICL gene expression is induced by the bacterial pathogen-associated molecular pattern (PAMP) flg22. T-DNA insertion alleles of PICC, but not PICL, show increased susceptibility to the non-virulent strain P. syringae pv. tomato DC3000 hrcC, but not to the virulent strain P. syringae pv. tomato DC3000. This suggests that PICC mutants are compromised in PAMP-triggered immunity (PTI). The data presented here provide first evidence for the involvement of a plant long coiled-coil protein in a plant defense response.
PMCID: PMC3581476  PMID: 23451199
2.  Novel plant SUN–KASH bridges are involved in RanGAP anchoring and nuclear shape determination 
The Journal of Cell Biology  2012;196(2):203-211.
SUN–KASH nuclear envelope bridges formed by WIP and SUN proteins are present in the plant branch of the tree of life but have functionally diverged from their opisthokont counterparts and are involved in nuclear morphology and RanGAP–nuclear envelope association.
Inner nuclear membrane Sad1/UNC-84 (SUN) proteins interact with outer nuclear membrane (ONM) Klarsicht/ANC-1/Syne homology (KASH) proteins, forming linkers of nucleoskeleton to cytoskeleton conserved from yeast to human and involved in positioning of nuclei and chromosomes. Defects in SUN–KASH bridges are linked to muscular dystrophy, progeria, and cancer. SUN proteins were recently identified in plants, but their ONM KASH partners are unknown. Arabidopsis WPP domain–interacting proteins (AtWIPs) are plant-specific ONM proteins that redundantly anchor Arabidopsis RanGTPase–activating protein 1 (AtRanGAP1) to the nuclear envelope (NE). In this paper, we report that AtWIPs are plant-specific KASH proteins interacting with Arabidopsis SUN proteins (AtSUNs). The interaction is required for both AtWIP1 and AtRanGAP1 NE localization. AtWIPs and AtSUNs are necessary for maintaining the elongated nuclear shape of Arabidopsis epidermal cells. Together, our data identify the first KASH members in the plant kingdom and provide a novel function of SUN–KASH complexes, suggesting that a functionally diverged SUN–KASH bridge is conserved beyond the opisthokonts.
PMCID: PMC3265956  PMID: 22270916
3.  Identification and characterization of the Arabidopsis FG-repeat nucleoporin Nup62 
Plant Signaling & Behavior  2011;6(3):330-334.
Ran is a multifunctional small GTPase that is involved in nucleocytoplasmic transport, mitotic spindle assembly and nuclear envelope reformation. Nuclear transport factor 2 (NTF2) facilitates nuclear import of Ran. It binds FxFG repeat-containing domains of the nucleoporins Nup62 (vertebrate) and Nsp1p (yeast). Here, we have identified Arabidopsis Nup62 through its sequence similarity to mammalian Nup62 and yeast Nsp1p. A GFP-AtNup62 fusion protein is associated with the nuclear envelope in transgenic Arabidopsis plants and interacts in planta with AtNTF2a, one of the two Arabidopsis NTF2 homologs. Overexpression-based co-suppression of AtNup62 leads to severely dwarfed, early-flowering plants, suggesting an important function for Nup62 in plants.
PMCID: PMC3142410  PMID: 21673506
Arabidopsis; nucleoporin; nuclear import; Ran GTPase; Nup62; NTF2
4.  The Arabidopsis Nuclear Pore and Nuclear Envelope 
The nuclear envelope is a double membrane structure that separates the eukaryotic cytoplasm from the nucleoplasm. The nuclear pores embedded in the nuclear envelope are the sole gateways for macromolecular trafficking in and out of the nucleus. The nuclear pore complexes assembled at the nuclear pores are large protein conglomerates composed of multiple units of about 30 different nucleoporins. Proteins and RNAs traffic through the nuclear pore complexes, enabled by the interacting activities of nuclear transport receptors, nucleoporins, and elements of the Ran GTPase cycle. In addition to directional and possibly selective protein and RNA nuclear import and export, the nuclear pore gains increasing prominence as a spatial organizer of cellular processes, such as sumoylation and desumoylation. Individual nucleoporins and whole nuclear pore subcomplexes traffic to specific mitotic locations and have mitotic functions, for example at the kinetochores, in spindle assembly, and in conjunction with the checkpoints. Mutants of nucleoporin genes and genes of nuclear transport components lead to a wide array of defects from human diseases to compromised plant defense responses. The nuclear envelope acts as a repository of calcium, and its inner membrane is populated by functionally unique proteins connected to both chromatin and—through the nuclear envelope lumen—the cytoplasmic cytoskeleton. Plant nuclear pore and nuclear envelope research—predominantly focusing on Arabidopsis as a model—is discovering both similarities and surprisingly unique aspects compared to the more mature model systems. This chapter gives an overview of our current knowledge in the field and of exciting areas awaiting further exploration.
PMCID: PMC3244964  PMID: 22303264
5.  NUA Activities at the Plant Nuclear Pore 
Plant Signaling & Behavior  2007;2(6):553-555.
NUA (Nuclear Pore Anchor), the Arabidopsis homolog of Tpr (Translocated Promoter Region), is one of the few nuclear pore proteins conserved between animals, yeast and plants. In the May issue of Plant Cell, we report that null mutants of NUA show a pleiotropic, early flowering phenotype accompanied by changes in SUMo and RNA homeostasis. We have shown that the early flowering phenotype is caused by changed abundances of flowering time regulators involved in several pathways. Arabidopsis nua mutants phenocopy mutants lacking the ESD4 (EARlY IN ShoRT DAYS 4) SUMo protease, similar to mutants of their respective yeast homologs. however, in contrast to the comparable yeast mutants, ESD4 does not appear to be delocalized from the nuclear pore in nua mutants. Taken together, our experimental data suggests a role for NUA in controlling mRNA export from the nucleus as well as SUMo protease activity at the nuclear pore, comparable but not identical to its homologs in other eukaryotes. Furthermore, characterization of NUA illustrates a potential link at the nuclear pore between SUMo modification, RNA homeostasis and plant developmental control.
PMCID: PMC2634367  PMID: 19704557
nuclear pore complex; nucleoporin; nuclear envelope; nucleocytoplasmic transport; SUMO; mRNA export; flowering time
6.  Coiled-coil protein composition of 22 proteomes – differences and common themes in subcellular infrastructure and traffic control 
Long alpha-helical coiled-coil proteins are involved in diverse organizational and regulatory processes in eukaryotic cells. They provide cables and networks in the cyto- and nucleoskeleton, molecular scaffolds that organize membrane systems and tissues, motors, levers, rotating arms, and possibly springs. Mutations in long coiled-coil proteins have been implemented in a growing number of human diseases. Using the coiled-coil prediction program MultiCoil, we have previously identified all long coiled-coil proteins from the model plant Arabidopsis thaliana and have established a searchable Arabidopsis coiled-coil protein database.
Here, we have identified all proteins with long coiled-coil domains from 21 additional fully sequenced genomes. Because regions predicted to form coiled-coils interfere with sequence homology determination, we have developed a sequence comparison and clustering strategy based on masking predicted coiled-coil domains. Comparing and grouping all long coiled-coil proteins from 22 genomes, the kingdom-specificity of coiled-coil protein families was determined. At the same time, a number of proteins with unknown function could be grouped with already characterized proteins from other organisms.
MultiCoil predicts proteins with extended coiled-coil domains (more than 250 amino acids) to be largely absent from bacterial genomes, but present in archaea and eukaryotes. The structural maintenance of chromosomes proteins and their relatives are the only long coiled-coil protein family clearly conserved throughout all kingdoms, indicating their ancient nature. Motor proteins, membrane tethering and vesicle transport proteins are the dominant eukaryote-specific long coiled-coil proteins, suggesting that coiled-coil proteins have gained functions in the increasingly complex processes of subcellular infrastructure maintenance and trafficking control of the eukaryotic cell.
PMCID: PMC1322226  PMID: 16288662
7.  MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure 
Nucleic Acids Research  2003;31(17):5175-5185.
Plastid DNA, like bacterial and mitochondrial DNA, is organized into protein–DNA complexes called nucleoids. Plastid nucleoids are believed to be associated with the inner envelope in developing plastids and the thylakoid membranes in mature chloroplasts, but the mechanism for this re-localization is unknown. Here, we present the further characterization of the coiled-coil DNA-binding protein MFP1 as a protein associated with nucleoids and with the thylakoid membranes in mature chloroplasts. MFP1 is located in plastids in both suspension culture cells and leaves and is attached to the thylakoid membranes with its C-terminal DNA-binding domain oriented towards the stroma. It has a major DNA-binding activity in mature Arabidopsis chloroplasts and binds to all tested chloroplast DNA fragments without detectable sequence specificity. Its expression is tightly correlated with the accumulation of thylakoid membranes. Importantly, it is associated in vivo with nucleoids, suggesting a function for MFP1 at the interface between chloroplast nucleoids and the developing thylakoid membrane system.
PMCID: PMC212795  PMID: 12930969
8.  Four signature motifs define the first class of structurally related large coiled-coil proteins in plants. 
BMC Genomics  2002;3:9.
Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants.
We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP) were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms.
Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom.
PMCID: PMC102765  PMID: 11972898

Results 1-8 (8)