Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.
- MeSH
- Arabidopsis cytology genetics metabolism MeSH
- Brefeldin A pharmacology MeSH
- Cell Membrane metabolism MeSH
- Cytokinins chemistry metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Fluorescent Dyes chemistry metabolism MeSH
- Plants, Genetically Modified MeSH
- Meristem cytology metabolism MeSH
- Protein Kinases genetics metabolism MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Receptors, Cell Surface genetics metabolism MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Signal Transduction drug effects MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.
- MeSH
- Endoplasmic Reticulum metabolism MeSH
- Phosphatidylglycerols metabolism MeSH
- Type C Phospholipases metabolism MeSH
- Homeostasis MeSH
- Lipid Droplets chemistry MeSH
- Lipid Metabolism MeSH
- Mitochondria metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae enzymology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.
- MeSH
- Acrylamides pharmacology MeSH
- Active Transport, Cell Nucleus MeSH
- Cell Nucleus metabolism MeSH
- 3T3-L1 Cells MeSH
- Hep G2 Cells MeSH
- Cytoplasm metabolism MeSH
- Histones metabolism MeSH
- Cell Cycle Checkpoints MeSH
- Humans MeSH
- Mutagenesis, Site-Directed MeSH
- Mice MeSH
- NAD metabolism MeSH
- Nicotinamide Phosphoribosyltransferase chemistry genetics metabolism MeSH
- Oxidative Stress MeSH
- Piperidines pharmacology MeSH
- Poly(ADP-ribose) Polymerases metabolism MeSH
- Cell Proliferation MeSH
- Recombinant Fusion Proteins chemistry genetics metabolism MeSH
- Sirtuins metabolism MeSH
- Cell Survival drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.
- MeSH
- Arabidopsis drug effects genetics microbiology MeSH
- Botrytis drug effects physiology MeSH
- Fluorescence MeSH
- Plants, Genetically Modified MeSH
- Plant Roots drug effects genetics MeSH
- RNA, Messenger genetics metabolism MeSH
- Plant Diseases genetics microbiology MeSH
- Disease Resistance drug effects genetics MeSH
- Peptides pharmacology MeSH
- Proteolysis drug effects MeSH
- Gene Expression Regulation, Plant drug effects MeSH
- Plant Proteins genetics metabolism MeSH
- Seedlings drug effects growth & development physiology MeSH
- Solanum lycopersicum microbiology MeSH
- Subcellular Fractions metabolism MeSH
- RNA Transport drug effects genetics MeSH
- Plant Shoots drug effects genetics MeSH
- Green Fluorescent Proteins metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Mediator is a multiprotein complex that connects regulation mediated by transcription factors with RNA polymerase II transcriptional machinery and integrates signals from the cell regulatory cascades with gene expression. One of the Mediator subunits, Mediator complex subunit 28 (MED28), has a dual nuclear and cytoplasmic localization and function. In the nucleus, MED28 functions as part of Mediator and in the cytoplasm, it interacts with cytoskeletal proteins and is part of the regulatory cascades including that of Grb2. MED28 thus has the potential to bring cytoplasmic regulatory interactions towards the centre of gene expression regulation. In this study, we identified MDT-28, the nematode orthologue of MED28, as a likely target of lysine acetylation using bioinformatic prediction of posttranslational modifications. Lysine acetylation was experimentally confirmed using anti-acetyl lysine antibody on immunoprecipitated GFP::MDT-28 expressed in synchronized C. elegans. Valproic acid (VPA), a known inhibitor of lysine deacetylases, enhanced the lysine acetylation of GFP::MDT-28. At the subcellular level, VPA decreased the nuclear localization of GFP::MDT-28 detected by fluorescencelifetime imaging microscopy (FLIM). This indicates that the nuclear pool of MDT-28 is regulated by a mechanism sensitive to VPA and provides an indirect support for a variable relative proportion of MED28 orthologues with other Mediator subunits.
- MeSH
- Acetylation MeSH
- Cell Nucleus drug effects metabolism MeSH
- Caenorhabditis elegans drug effects metabolism MeSH
- Densitometry MeSH
- Nuclear Proteins chemistry metabolism MeSH
- Valproic Acid pharmacology MeSH
- Larva drug effects MeSH
- Humans MeSH
- Lysine metabolism MeSH
- Mediator Complex chemistry metabolism MeSH
- Caenorhabditis elegans Proteins chemistry metabolism MeSH
- Recombinant Fusion Proteins metabolism MeSH
- Amino Acid Sequence MeSH
- Sequence Homology, Amino Acid * MeSH
- Protein Transport drug effects MeSH
- Computational Biology MeSH
- Green Fluorescent Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.
- MeSH
- Arabidopsis enzymology immunology microbiology MeSH
- Phosphatidylcholines metabolism MeSH
- Type C Phospholipases physiology MeSH
- Golgi Apparatus enzymology MeSH
- Plant Immunity physiology MeSH
- Cloning, Molecular MeSH
- Microscopy, Confocal MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Plant Diseases immunology microbiology MeSH
- Arabidopsis Proteins physiology MeSH
- Protoplasts enzymology MeSH
- Pseudomonas syringae * MeSH
- Reactive Oxygen Species MeSH
- Gene Expression Regulation, Plant MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.
- MeSH
- Arabidopsis metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Endosomes metabolism MeSH
- Phosphatidylinositol Phosphates metabolism MeSH
- Golgi Apparatus metabolism MeSH
- Microscopy, Confocal MeSH
- Plant Leaves metabolism MeSH
- Promoter Regions, Genetic MeSH
- Arabidopsis Proteins genetics metabolism MeSH
- Recombinant Fusion Proteins biosynthesis genetics MeSH
- Nicotiana metabolism MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Publication type
- Journal Article MeSH
Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies.
- MeSH
- Glycosylation MeSH
- Microscopy, Confocal MeSH
- Humans MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae genetics metabolism MeSH
- Subcellular Fractions MeSH
- Tetraspanins genetics metabolism MeSH
- Protein Transport MeSH
- Green Fluorescent Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.
- MeSH
- Cell Membrane metabolism MeSH
- Fungal Proteins metabolism MeSH
- Microscopy, Confocal MeSH
- Real-Time Polymerase Chain Reaction MeSH
- NADPH Oxidases genetics metabolism MeSH
- Phytophthora physiology MeSH
- Reactive Oxygen Species metabolism MeSH
- Plant Proteins genetics metabolism MeSH
- Nicotiana genetics metabolism microbiology MeSH
- Microscopy, Electron, Transmission MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
TPX2 performs multiple roles in microtubule organization. Previously, it was shown that plant AtTPX2 binds AtAurora1 kinase and colocalizes with microtubules in a cell cycle-specific manner. To elucidate the function of TPX2 further, this work analysed Arabidopsis cells overexpressing AtTPX2-GFP. Distinct arrays of bundled microtubules, decorated with AtTPX2-GFP, were formed in the vicinity of the nuclear envelope and in the nuclei of overexpressing cells. The microtubular arrays showed reduced sensitivity to anti-microtubular drugs. TPX2-mediated formation of nuclear/perinuclear microtubular arrays was not specific for the transition to mitosis and occurred independently of Aurora kinase. The fibres were not observed in cells with detectable programmed cell death and, in this respect, they differed from TPX2-dependent microtubular assemblies functioning in mammalian apoptosis. Colocalization and co-purification data confirmed the interaction of importin with AtTPX2-GFP. In cells with nuclear foci of overexpressed AtTPX2-GFP, strong nuclear signals for Ran and importin diminished when microtubular arrays were assembled. This observation suggests that TPX2-mediated microtubule formation might be triggered by a Ran cycle. Collectively, the data suggest that in the acentrosomal plant cell, in conjunction with importin, overexpressed AtTPX2 reinforces microtubule formation in the vicinity of chromatin and the nuclear envelope.
- MeSH
- Apoptosis MeSH
- Arabidopsis cytology enzymology metabolism MeSH
- Cell Nucleus metabolism MeSH
- Centrosome metabolism MeSH
- Chromatin metabolism MeSH
- Nuclear Envelope metabolism MeSH
- Karyopherins metabolism MeSH
- Aurora Kinases metabolism MeSH
- Microtubules metabolism MeSH
- Mitosis MeSH
- Computer Simulation MeSH
- Microtubule-Associated Proteins metabolism MeSH
- Arabidopsis Proteins metabolism MeSH
- Plant Cells metabolism MeSH
- Subcellular Fractions metabolism MeSH
- Protein Transport MeSH
- Tubulin metabolism MeSH
- Green Fluorescent Proteins metabolism MeSH
- Imaging, Three-Dimensional MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
