• Korean Journal of Microbiology
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• v.52 no.3
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• pp.383-387
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• 2016

In fission yeast, Schizosaccharomyces pombe, the cdc31 gene encodes a member of the conserved $Ca^{2+}$-binding centrin/CDC31 family, which is a component of spindle pole body. Here, we demonstrate that the S. pombe cdc31p is also a component of TREX-2 complex, which influences mRNA export from the nucleus to the cytoplasm. Repression of the cdc31 gene expression caused growth defect with accumulation of $poly(A)^+$ RNA in the nucleus. On the other hand, over-expression of cdc31 exhibited no defects of both growth and bulk mRNA export, but showed somewhat longer cell morphology. Yeast two-hybrid analysis showed that Cdc31 interacted with Sac3 and Pci2, the subunits of TREX-2 complex. These results suggest that S. pombe Cdc31 is also involved in mRNA export as a component of TREX-2 complex.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.112-116
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• 2019

Thp1/PCID2 is a subunit of the evolutionally conserved TREX-2 complex, which is required for transcription-coupled mRNA export from the nucleus to the cytoplasm. In fission yeast, Schizosaccharomyces pombe, there are two orthologs of the Thp1/PCID2 protein. In addition to pci2 (SPBC1105.07c) gene, SPAC1B3.08 gene encodes a PCI domain-containing protein that is predicted as a component of TREX-2 complex. Overexpression of SPAC1B3.08 cause slight defects of both growth and mRNA export. Yeast two-hybrid and co-immunoprecipitation analysis exhibits that the SPAC1B3.08 protein interacted with Sac3 and Dss1, which are another components of TREX-2 complex. These observations support the possibility that the S. pombe SPAC1B3.08 protein, as a component of TREX-2 complex, is involved in mRNA export.

• Journal of Life Science
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• v.22 no.12
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• pp.1608-1613
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• 2012

A conventional kinesin, KIF5/Kinesin-I, transports various cargoes along the microtubule through interaction between its light chain subunit and the cargoes. Kinesin light chains (KLCs) interact with many different cargoes using their tetratricopeptide repeat (TPR) domain, but the mechanism underlying recognition and binding of a specific cargo has not yet been completely elucidated. We used the yeast two-hybrid assay to identify proteins that interact with the TPR domain of KLC1. We found an interaction between the TPR domain of KLC1 and an amyloid precursor protein (APP)-binding protein PAT1 (protein interacting with APP tail 1). The yeast two-hybrid assay demonstrated that the TPR domain-containing region of KLC1 mediated binding to the C-terminal tail region of PAT1. PAT1 also bound to KLC2 but not to kinesin heavy chains (KIF5A, KIF5B, and KIF5C) in the yeast two-hybrid assay. These protein-protein interactions were also observed in the glutathione S-transferase (GST) pull-down assay and by co-immunoprecipitation. Anti-PAT1 antibody as well as anti-APP anti-body co-immunoprecipitated KLC and KHCs associated with PAT1 from mouse brain extracts. These results suggest that PAT1 could mediate interactions between Kinesin-I and APP containing vesicles.

• Journal of Life Science
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• v.21 no.8
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• pp.1076-1082
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• 2011

Microtubule-based kinesin motor proteins are used for long-range vesicular transport. KIF5s (KIF5A, KIF5B and KIF5C) mediate the transport of various membranous vesicles along microtubules, but the mechanism behind how they recognize and bind to a specific cargo has not yet been completely elucidated. To identify the interaction protein for KIF5B, yeast two-hybrid screening was performed and a specific interaction with the unconventional myosin Myo9b, an actin-based vesicle transport motor, was found. The GTPase-activating protein (GAP) domain of Myo9s was essential for interaction with KIF5B in the yeast two-hybrid assay. Myo9b bound to the carboxyl-terminal region of KIF5B and to other KIF5 members. In addition, glutathione S-transferase (GST) pull-downs showed that Myo9s specifically interact to the complete Kinesin-I complex. An antibody to KIF5B specifically co-immunoprecipitated KIF5B associated with Myo9s from mouse brain extracts. These results suggest that kinesin-I motor protein interacts directly with actin-based motor proteins in the cell.

• Journal of Life Science
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• v.19 no.7
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• pp.859-865
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• 2009

Microtubules, a major cytoskeleton, form parallel arrays in the axon and are oriented with their plus ends toward the cell periphery. Kinesin superfamily proteins (KIFs) are the molecular motors acting in the microtubule-based motilities of organelles in cells. Here, we used the yeast two-hybrid system to identify the protein that interacts with the coiled-coil domain of KIF1A and found a specific interaction with microtubule-destabilizing factor SCG10. SCG10 bound to the amino acid residues between 400 and 820 of KIF1A, but not to other KIFs in the yeast two-hybrid assay. The coiled-coil domain of SCG10 is essential for interaction with KIF1A. In addition, this specific interaction was also observed in the Glutathione S-transferase pull-down assay. An antibody to SCG10 specifically co-immunoprecipitated KIF1A associated with SCG10 from mouse brain extracts. These results suggest that KIF1A motor protein transports SCG10-containing vesicles along microtubules in neurons.

• Journal of Applied Biological Chemistry
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• v.55 no.1
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• pp.67-70
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• 2012

Plant MYB transcription factors regulate secondary metabolism, cellular morphogenesis, and plant hormone signaling pathway. MYB proteins in plants consist of two repeats of 50 amino acid residues, which are referred to as R2R3 and they interact with WD40 or basic helix loop helix (bHLH) proteins. Yeast two hybrid assay was determined whether rice MYB protein interacts with either OsTTG1, which contains a WD40 domain, or with OsGL3, which contains a bHLH domain. Among 30 OsMYB proteins, three interacted with OsTTG1 and five interacted with OsGL3. A series of MYB mutants were created to determine the MYB domain important for the interaction with OsTTG1 or OsGL3. By using the yeast two hybrid assay, we found that the R3 motif of OsMYB10 and the R2 motif of OsMYB16 were required for interaction with OsTTG1 and OsGL3 proteins, respectively.

• Journal of Life Science
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• v.27 no.6
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• pp.673-679
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• 2017

Microtubules are long rods in the cytoplasm of cells that plays a role in cell motility and intracellular transport. Microtubule-based transport by motor proteins is essential in intracellular transport. Kinesin 1 is a molecular motor protein that mediates the intracellular transport of various membranous vesicles, mRNAs, and proteins along microtubules. It is comprised of two heavy chains (KHCs, also called KIF5s) and two light chains (KLCs). KIF5s bear a motor domain in their amino (N)-terminal regions and interact with various cargoes through the cargo-binding domain in their carboxyl (C)-terminal regions. To identify proteins interacting with KIF5B, yeast two-hybrid screening was performed, and a specific interaction with the cytoplasmic linker protein 170 (CLIP-170), a plus end microtubule-binding protein, was found. The coiled-coil domain of CLIP-170 is essential for interactions with KIF5B in the yeast two-hybrid assay. CLIP-170 bound to the cargo-binding domain of KIF5B. Also, other KIF5s, KIF5A and KIF5C, interacted with CLIP-170 in the yeast two-hybrid assay. In addition, glutathione S-transferase (GST) pull-downs showed that KIF5s specifically interacted with CLIP-170. An antibody to KIF5B specifically co-immunoprecipitated CLIP-170 associated with KIF5B from mouse brain extracts. These results suggest that kinesin 1 motor protein may transport CLIP-170 in cells.

• Parasites, Hosts and Diseases
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• v.44 no.4 s.140
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• pp.303-312
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• 2006

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the $X-\alpha-gal$ positive and PCR, 157 colonies of the $X-\beta-gal$ assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

• Korean Journal of Microbiology
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• v.51 no.2
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• pp.181-185
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• 2015

Tho2/THOC2 is a subunit of the THO complex that plays important roles in mRNP biogenesis connecting transcription with mRNA maturation and export. A fission yeast, Schizosaccharomyces pombe, ortholog of Tho2/THOC2 was identified from the genome database. Tetrad analysis showed that the S. pombe tho2 is essential for growth. Repression or overexpression of the tho2 gene caused growth defect with elongated cells, abnormal DNA distribution, and accumulation of $poly(A)^+$ RNA in the nucleus. And the functional GFP-Tho2 protein is localized mainly in the nucleus. Yeast two-hybrid analysis showed that Tho2 interacted with Tex1, another subunit of THO complex. These results suggest that S. pombe Tho2 is also involved in mRNA export from the nucleus and is a component of THO complex.

• Proceedings of the Korean Aquaculture Society Conference
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• 2004.05a
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• pp.199-200
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• 2004