• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.472-476
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• 2008

Two isoforms of human CD99 have been identified, but only heterotypic interaction between the isomers was recently demonstrated. In this study, we performed bimolecular fluorescence complementation analysis to further characterize the interaction in vivo. Upon transiently transfecting plasmids expressing either of the two isoforms fused with yellow fluorescent protein (YFP) fragments, all the YFP-tagged CD99 molecules were properly localized on cell surfaces, and formed fluorescent dimers. Interestingly, however, unlike the previous report, the homodimers formed as efficiently as the heterodimer via their extracellular domains, implying its distinct regulatory role through modulating the complex profile.

• The Plant Pathology Journal
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• 제29권4호
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• pp.454-459
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• 2013

The Potexvirus Alternanthera mosaic virus (AltMV) has multifunctional triple gene block (TGB) proteins, among which our studies have focused on the properties of the TGB1 protein. The TGB1 of AltMV has functions including RNA binding, RNA silencing suppression, and cell-to-cell movement, and is known to form homologous interactions. The helicase domains of AltMV TGB1 were separately mutated to identify which regions are involved in homologous TGB1 interactions. The yeast two hybrid system and Bimolecular Fluorescence Complementation (BiFC) in planta were utilized to examine homologous interactions of the mutants. Helicase motif I of AltMV TGB1 was found to be critical to maintain homologous interactions. Mutations in the remaining helicase motifs did not inhibit TGB1 homologous interactions. In the absence of homologous interaction of TGB1, subcellular localization of helicase domain I mutants showed distinctively different patterns from that of WT TGB1. These results provide important information to study viral movement and replication of AltMV.

• BMB Reports
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• 제49권10호
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• pp.542-547
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• 2016

Acid-sensing ion channels (ASICs) are proton-gated cation channels widely expressed in the nervous system. Proton sensing by ASICs has been known to mediate pain, mechanosensation, taste transduction, learning and memory, and fear. In this study, we investigated the differential subcellular localization of ASIC2a and ASIC3 in heterologous expression systems. While ASIC2a targeted the cell surface itself, ASIC3 was mostly accumulated in the ER with partial expression in the plasma membrane. However, when ASIC3 was co-expressed with ASIC2a, its surface expression was markedly increased. By using bimolecular fluorescence complementation (BiFC) assay, we confirmed the heteromeric association between ASIC2a and ASIC3 subunits. In addition, we observed that the ASIC2a-dependent surface trafficking of ASIC3 remarkably enhanced the sustained component of the currents. Our study demonstrates that ASIC2a can increase the membrane conductance sensitivity to protons by facilitating the surface expression of ASIC3 through herteromeric assembly.

• Molecules and Cells
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• 제26권3호
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• pp.270-277
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• 2008

This study addresses the physiological functions of the Ran-binding protein homolog NbRanBP1 in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbRanBP1 caused stunted growth, leaf yellowing, and abnormal leaf morphology. The NbRanBP1 gene was constitutively expressed in diverse tissues and an NbRanBP1:GFP fusion protein was primarily localized to the nuclear rim and the cytosol. BiFC analysis revealed in vivo interaction between NbRanBP1 and NbRan1 in the nuclear envelope and the cytosol. Depletion of NbRanBP1 or NbRan1 reduced nuclear accumulation of a NbBTF3:GFP marker protein. In the later stages of development, NbRanBP1 VIGS plants showed stress responses such as reduced mitochondrial membrane potential, excessive production of reactive oxygen species, and induction of defense-related genes. The molecular role of RanBP1 in plants is discussed in comparison with RanBP1 function in yeast and mammals.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.149-149
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• 2017

The RING (Really Interesting New Gene) finger proteins are known to play crucial roles in various abiotic stresses in plants. In this study, we report on RING finger E3 ligase, ${\underline{O}ryza}$ ${\underline{s}ativa}$ ${\underline{s}alt$-, ${\underline{A}BA}$- and ${\underline{d}rounght}$ stress-${\underline{i}nduced}$ RING finger ${\underline{p}}rotein{\underline{1}}$ gene (OsSAD1). In vitro ubiquitination assay demonstrated that unlike OsSAD1, a single amino acid substitution ($OsSAD1^{C168A}$) of the RING domain showed no E3 ligase activity, supporting the notion that the activity of most E3s is specified by a RING domain. Result of Yeast-Two hybridization, In vivo protein degradation assay supports that OsSAD1 interacting with 3 substrate, OsSNAC2, OsGRAS44 and OsPIRIN1, and mediates proteolysis of 3 substrates via the 26S proteasome pathway. Subcellular localizations of OsSAD1 while approximately 62% of transient signals were detected in cytosol, 38% of signals were showed nucleus. However, transiently expression of OsSAD1 was detected in cytosol 30% while as 70% of nucleus under 200 mM salt treated rice protoplasts. Results of bimolecular fluorescence complementation (BiFC) showed that two nucleus-localized proteins (OsSNAC2 and OsGRAS44) interacted with OsSAD1 in the both cytosol and nucleus. Heterogeneous overexpression of OsSAD1 Heterogeneous overexpresssion of OsSAD1 in Arabidopsis exhibited sensitive phenotypes with respect to Salt-, mannitol-responsive seed germination, seedling growth. In ABA conditions, OsSAD1 overexpression plants showed highly tolerance phenotypes, such as root length and stomatal closure. Our findings suggest that the OsSAD1 may play a negative regulator in salt stress response by modulating levels of its target proteins.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.90-90
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• 2017

Regulation of seed dormancy is important in many grains to prevent pre-harvest sprouting. To identify and understand the gene related to seed dormancy regulation, we have screened for viviparous phenotypes of rice mutant lines generated by insertion of Ds transposon in a Korean Japonica cultivar (Dongjin) background. One of the mutants, which represented viviparous phenotype, was selected for further seed dormancy regulation studies and designated dor1. The dor1 mutant has single Ds insertion in the second exon of OsDor1 gene encoding glycine-rich protein. The seeds of dor1 mutant showed a higher germination potential and reduced abscisic acid (ABA) sensitivity compared to wild type Dongjin. Over-expression of Dor1 complements the viviparous phenotype of dor1 mutant, indicating that Dor1 function in seed dormancy regulation. Subcellular localization assay of Dor1-GFP fusion protein revealed that the OsDor1 protein mainly localized to membrane and the localization of OsDOR1 was influenced by presence of a giberelin (GA) receptor OsGID1. Further bimolecular fluorescence complementation (BiFC) analysis indicated that OsDOR1 interact with OsGID1. The combined results suggested that OsDOR1 regulates seed dormancy by interacting with OsGID1 in GA response. Additionally, expression of OsDOR1 partially complemented the cold sensitivity of Escherichia coli BX04 mutant lacking four cold shock proteins, indicating that OsDOR1 possessed RNA chaperone activity.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.357-364
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• 2017

In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

• Molecules and Cells
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• 제38권1호
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• pp.81-88
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• 2015

Flowering time (or heading date) is controlled by intrinsic genetic programs in response to environmental cues, such as photoperiod and temperature. Rice, a facultative short-day (SD) plant, flowers early in SD and late in long-day (LD) conditions. Casein kinases (CKs) generally act as positive regulators in many signaling pathways in plants. In rice, Heading date 6 (Hd6) and Hd16 encode $CK2{\alpha}$ and CKI, respectively, and mainly function to delay flowering time. Additionally, the major LD-dependent floral repressors Hd2/Oryza sativa Pseudo-Response Regulator 37 (OsPRR37;hereafter PRR37) and Ghd7 also confer strong photoperiod sensitivity. In floral induction, Hd16 acts upstream of Ghd7 and CKI interacts with and phosphorylates Ghd7. In addition, Hd6 and Hd16 also act upstream of Hd2. However, whether CKI and $CK2{\alpha}$ directly regulate the function of PRR37 remains unclear. Here, we use in vitro pull-down and in vivo bimolecular fluorescence complementation assays to show that CKI and $CK2{\alpha}$ interact with PRR37. We further use in vitro kinase assays to show that CKI and $CK2{\alpha}$ phosphorylate different regions of PRR37. Our results indicate that direct posttranslational modification of PRR37 mediates the genetic interactions between these two protein kinases and PRR37. The significance of CK-mediated phosphorylation for PRR37 and Ghd7 function is discussed.

• Molecules and Cells
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• 제45권6호
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• pp.413-424
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• 2022

Suppressor of mothers against decapentaplegic homolog (SMAD) 4 is a pluripotent signaling mediator that regulates myriad cellular functions, including cell growth, cell division, angiogenesis, apoptosis, cell invasion, and metastasis, through transforming growth factor β (TGF-β)-dependent and -independent pathways. SMAD4 is a critical modulator in signal transduction and functions primarily as a transcription factor or cofactor. Apart from being a DNA-binding factor, the additional SMAD4 mechanisms in tumor suppression remain elusive. We previously identified methyl malonyl aciduria cobalamin deficiency B type (MMAB) as a critical SMAD4 binding protein using a proto array analysis. This study confirmed the interaction between SMAD4 and MMAB using bimolecular fluorescence complementation (BiFC) assay, proximity ligation assay (PLA), and conventional immunoprecipitation. We found that transient SMAD4 overexpression down-regulates MMAB expression via a proteasome-dependent pathway. SMAD4-MMAB interaction was independent of TGF-β signaling. Finally, we determined the effect of MMAB downregulation on cancer cells. siRNA-mediated knockdown of MMAB affected cancer cell metabolism in HeLa cells by decreasing ATP production and glucose consumption as well as inducing apoptosis. These findings suggest that SMAD4 controls cancer cell metabolism by regulating MMAB.

• Molecules and Cells
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• 제38권3호
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• pp.259-266
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• 2015

The regulation of flowering time has crucial implications for plant fitness. MicroRNA156 (miR156) represses the floral transition in Arabidopsis thaliana, but the mechanisms regulating its transcription remain unclear. Here, we show that two AGAMOUS-like proteins, AGL15 and AGL18, act as positive regulators of the expression of MIR156. Small RNA northern blot analysis revealed a significant decrease in the levels of mature miR156 in agl15 agl18 double mutants, but not in the single mutants, suggesting that AGL15 and AGL18 co-regulate miR156 expression. Histochemical analysis further indicated that the double mutants showed a reduction in MIR156 promoter strength. The double mutants also showed reduced abundance of pri-miR156a and pri-miR156c, two of the primary transcripts from MIR156 genes. Electrophoretic mobility shift assays demonstrated that AGL15 directly associated with the CArG motifs in the MIR156a/c promoters. AGL18 did not show binding affinity to the CArG motifs, but pull-down and yeast two-hybrid assays showed that AGL18 forms a heterodimer with AGL15. GFP reporter assays and bimolecular fluorescence complementation (BiFC) showed that AGL15 and AGL18 co-localize in the nucleus and confirmed their in vivo interaction. Overexpression of miR156 did not affect the levels of AGL15 and AGL18 transcripts. Taking these data together, we present a model for the transcriptional regulation of MIR156. In this model, AGL15 and AGL18 may form a complex along with other proteins, and bind to the CArG motifs of the promoters of MIR156 to activate the MIR156 expression.