PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-4 (4)
 

Clipboard (0)
None

Select a Filter Below

Journals
Authors
more »
Year of Publication
Document Types
1.  BEX1/ARF1A1C is Required for BFA-Sensitive Recycling of PIN Auxin Transporters and Auxin-Mediated Development in Arabidopsis 
Plant and Cell Physiology  2014;55(4):737-749.
Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1–green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.
doi:10.1093/pcp/pct196
PMCID: PMC3982122  PMID: 24369434
Arabidopsis thaliana; Auxin; Embryogenesis; Exocytosis; PIN-FORMED
2.  Cell Polarity and Patterning by PIN Trafficking through Early Endosomal Compartments in Arabidopsis thaliana 
PLoS Genetics  2013;9(5):e1003540.
PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.
Author Summary
Auxin is a unique plant hormone, which is actively and directionally transported in plant tissues. Transported auxin locally accumulates in the plant body and triggers a multitude of responses, including organ formation and patterning. Therefore, regulation of the directional auxin transport is very important in multiple aspects of plant development. The PIN-FORMED (PIN) family of auxin transporters is known to localize at specific sides of cells and export auxin from the cells, enabling the directional transport of auxin in the tissues. PIN proteins are rapidly shuttling between the plasma membrane and intracellular compartments, potentially allowing dynamic changes of the asymmetric localization according to developmental and environmental cues. Here, we discovered that a mutation in the Sec1/Munc18 family protein VPS45 abolishes its own early endosomal localization and compromises intracellular trafficking of PIN proteins. By genetic and pharmacological inhibition of early endosomal trafficking, we also revealed that another early endosomal protein, ARF GEF BEN1, is involved in early endosomal trafficking at a distinct step. Furthermore, we showed that these components play crucial roles in polar localization and dynamic repolarization of PIN proteins, which underpin various developmental processes. These findings highlight the indispensable roles of early endosomal components in regulating PIN polarity and plant architecture.
doi:10.1371/journal.pgen.1003540
PMCID: PMC3667747  PMID: 23737757
3.  Auxin-inducible protein depletion system in fission yeast 
BMC Cell Biology  2011;12:8.
Background
Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe.
Results
We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest.
Conclusion
Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.
doi:10.1186/1471-2121-12-8
PMCID: PMC3048574  PMID: 21314938
4.  Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana 
Plant Signaling & Behavior  2011;6(1):66-71.
Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.
doi:10.4161/psb.6.1.13999
PMCID: PMC3122008  PMID: 21301212
cytokinin; cytokinin receptor; female gametophyte; male gametophyte; stigma

Results 1-4 (4)