• Journal of Microbiology
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• 제38권2호
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• pp.113-116
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• 2000

Escherichia coli F plasmid partition apparatus is composed of two trans-acting proteins (SopA and SopB) and one cis-acting DNA sequence (sopC). The SopB-sopC complex has been suggested to serve a centromere-like function through its interaction with chromosomally encoded proteins which remain to be identified. In this paper, we are introducing a new yeast 1.5-hybrid system which assembles the two-hybrid and one-hybrid system as a mean to find and additional component of the F plasmid partition system, interacting with DNA (sopC)-bound SopB protein. The results indicates that this system is a promising one, capable of selecting an interacting component.

• The Plant Pathology Journal
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• 제23권4호
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• pp.281-286
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• 2007

Interactions between viral proteins and host proteins are essential for virus replication. Especially, translation of viral genes completely depends on the host machinery. In potyviruses, interactions of genome-linked viral protein (VPg) with host translation factors including eIF4E, eIF(iso)4E, and poly(A)-binding protein (PABP) has previously been characterized. In this study, we investigated interactions between Soybean mosaic virus (SMV) viral proteins and host translation factors by yeast two-hybrid system. SMV VPg interacted with eIF4E, eIF(iso)4E, and PABP in yeast two-hybrid system, while SMV helper component proteinase (HC-pro) interacted with neither of those proteins. The interaction between SMV NIb and PABP was also detected. These results are consistent with those reported previously in other potyviruses. Interestingly, we found reproducible and specific interactions between SMV coat protein (CP) and PABP. Deletion analysis showed that the region of CP comprising amino acids 116 to 206 and the region of PABP comprising amino acids 520 to 580 are involved in CP/PABP interactions. Soybean library screening with SMV NIb by yeast two-hybrid assay also identified several soybean proteins including chlorophyll a/b binding preprotein, photo-system I-N subunit, ribulose 1,5-biphosphate carboxylase, ST-LSI protein, translation initiation factor 1, TIR-NBS type R protein, RNA binding protein, ubiquitin, and LRR protein kinase. Altogether, these results suggest that potyviral replicase may comprise a multi-protein complex with PABP, CP, and other host factors.

• BMB Reports
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• 제33권1호
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• pp.59-62
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• 2000

Hepatitis C virus (HCV) nonstructural 5B (NS5B) protein is an RNA-dependent RNA polymerase (RdRp). To determine whether it can contribute to viral replication by interaction with cellular proteins, the yeast two-hybrid screening system was employed to screen a human liver cDNA library. Using the HCV NS5B as a bait, we have isolated positive clones encoding a cellular protein. The NS5B interacting protein, 5BIP, is a novel cellular protein of 170 amino acids. Interaction of the HCV NS5B protein with 5BIP was confirmed by a protein-protein blotting assay. Recently, we have demonstrated that NS5B possesses an RdRp activity and thus it is possible that 5BIP, in association with NS5B, plays a role in HCV replication.

• 생명과학회지
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• 제22권12호
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• pp.1608-1613
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• 2012

KIF5/Kinesin-I는 경쇄(light chain)를 통하여 결합함으로써 다양한 운반체들을 미세소관을 따라 운반한다. Kinesin light chains (KLCs)은 tetratricopeptide repeat (TPR) 영역을 매개로 운반체와 결합한다. 현재까지 KLCs와 결합하는 많은 운반체들이 확인되었으나 KLCs가 어떻게 특정운반체를 인식하여 결합하는지는 아직 확실히 밝혀지지 않았다. 본 연구에서 KLC1의 TPR 영역과 결합하는 단백질을 분리하기 위하여 효모 two-hybrid system을 이용하여 탐색한 결과 amyloid precursor protein (APP)과 결합하는 것으로 보고된 protein interacting with APP tail 1 (PAT1)을 분리하였다. KLC1은 PAT1의 C-말단 부위와 결합하며, PAT1은 KLC1의 TPR 영역을 포함한 부위와 결합함을 효모 two-hybrid assay로 확인하였다. 또한 PAT1는 KLC2와도 결합하였지만 kinesin heavy chains (KHCs)인 KIF5A, KIF5B, KIF5C와는 결합하지 않았다. 단백질간 결합은 glutathione S-transferase (GST) pull-down assay와 공동면역침강으로도 확인하였다. 생쥐의 뇌 파쇄액을 PAT1 항체와 APP 항체로 면역침강을 행한 결과 KLC와 KHCs가 같이 침강하였다. 이러한 결과들은 PAT1이 Kinesin-I와 APP 포함 소포간의 상호작용을 매개한다는 것을 시사한다.

• 생명과학회지
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• 제17권7호통권87호
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• pp.889-895
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• 2007

KIF5는 2분자의 kinesin heavy chain (KHC)과 2분자의 kinesin light cham (KLC)으로 구성되며 미세소관과 직접 결합한다. KIF5는 여러가지 세포 내 소기관을 이동시키나 KIF5가 이동시키는 운반체가 어떻게 특이적으로 결합하는지는 아직 밝혀지지 못하였다. 본 연구에서 효모 two-hybrid system을 사용하여 KLC1의 tetratricopeptide repeats (TRP) 부위와 결합하는 세포 내의 단백질을 분리하였다. 결과 KLC1와 특이적으로 결합하는JNK/stress-activated protein kinase-associated protein 1 (JSAP1/JIP3)을 분리하였다. 이러한 결합은 KLC1의 TRP1, 2 영역과 JSAP1의 leucine zipper 영역이 결합에 관여하며, 또한 효모 two-hybrid assay에서 JSAP1은 KLC2와 결합하지만 신경세포에서 발현하는 KIF5A, KIF5C 그리고 모든 세포에서 발현하는 KIF5B와는 결합하지 않았다. 단백질간의 결합을 pull-down assay로 확인한 결과 KLC1은 glutathione S-transferase (GST)와는 결합하지 않으나 GST결한 JSAP1과는 결합하였다. 또한 생쥐의 뇌 파쇄 액으로부터 JSAP1 항체로 면역침강을 행한 결과 KLC1은 JSAP1과 같이 침강하였다. 이러한 결과들은 KLC1은 JSAP1과 결합하며, JSAP1은 KLC1의 수용체로세포 내 KIF5의 수송의 매개 단백질로 작용함을 시사한다.

• 생명과학회지
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• 제24권1호
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• pp.14-19
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• 2014

Kinesin-1은 2개의 장쇄(KHCs, 또는 KIF5s)와 2개의 단쇄(KLCs)가 결합한 복합체로 되어 있다. 본 연구에서 효모 two-hybrid system을 이용하여 중추신경계의 신경세포에서 주로 발현되는 KIF5A와 결합하는 단백질을 탐색한 결과 phosphatidylinositol-3,5-bisphosphate ($PI(3,5)P_2$)의 5번 위치 인산을 제거하는 탈인산화효소 Fig4(Sac3)를 분리하였다. KIF5A는 Fig4의 C-말단과 결합함을 효모 two-hybrid assay로 확인하였다. Fig4는 KIF5A의 C-말단과 결합하지만, 두 개의 다른 장쇄인 KIF5B와 KIF5C 그리고 KLC1와는 결합하지 않았다. 단백질 간 결합을 glutathione S-transferase pull-down assay와 공동면역침강으로 추가 검증하였다. 생쥐의 뇌 파쇄액을 KIF5A 항체로 면역 침강한 결과 Fig4가 같이 침강하였다. 이러한 결과들은 kinesin-1이 Fig4와 결합한 단백질 복합체 혹은 운반체를 세포 내에서 운반함을 시사한다.

• Animal cells and systems
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• 제6권4호
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• pp.317-322
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• 2002

To improve conventional yeast one-hybrid screening, we have developed an efficient mammalian one-hybrid system that allows rapid isolation of com-plementary DNAs which are able to induce human p14$^{ARF}$. tumor suppressor gene. A 1.5 kb promoter region of p14$^{ARF}$ was fused to EGFP to generate ARF promoter-EGFP reporter vector. This reporter plasmid was stably trans-fected into NIH3T3 cells for generation of reporter cell line. When the reporter cell line was infected with E2F-1 together with excess amounts of empty vector, the cells that received the positive modulator were readily identifiable by green fluorescence using FACS. The GFP-positive cells were cloned directly from the cultured cells and expanded in bulk culture. The genomic DNAs from GFP-positive cells were prepared and the CDNA insert in integrated retroviral genome was recovered by PCR using primers annealing to the retroviral vector sequences flanking the insert-cloning site. This system should be useful for efficient screening of expression CDNA libraries in mammalian cells to identify novel upstream regulators for spe-cific genes by one-hybrid interaction.ion.

• 생명과학회지
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• 제19권8호
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• pp.1055-1061
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• 2009

${\alpha}$-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) 수용체는 중추신경계에 널리 발현하며, 연접부위에서 글루타메이트에 의한 흥분성 신경전달의 역할을 행한다. 이러한 수용체의 조절은 학습과 기억에 관여한다. 본 연구에서는 AMPA receptor subunit glutamate receptor-interacting protein 1 (GRIP1)과 결합하는 단백질로 armadillo family인 p0071/plakophilin-4을 분리하였다. GRIPl 단백질은 p0071/plakophilin-4 단백질의 C-말단과 결합하며, armadillo family 단백질 중 p0071/plakophilin-4 과만 결합한다. 효모 two-hybrid assay에서 GRIPl의 Postsynaptic density-95/Discs large/Zona occludens-1 (PDZ) 영역이 p0071/plakophilin-4 단백질과의 결합에 관여하며, p0071/plakophilin-4 단백질의 C-말단 아미노산인 "S-X-V" 배열이 결합에 필수적이었다. 이러한 결합은 뇌 균질액에서 Glutathione S-transferase (GST) pull-down assay와 공면역침강법으로 확인하였다. 이러한 결과들은 AMPA 수용체가 연접후막에 안정적으로 위치하는 새로운 역할을 시사한다.

• 대한의생명과학회지
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• 제9권3호
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• pp.159-165
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• 2003

Ankyrins are a ubiquitously expressed family of intracellular adaptor proteins involved in targeting diverse proteins to specialized membrane domains in both the plasma membrane and the endoplasmic reticulum. Recently, the studies with C-terminus of ankyrins have identified that ankyrin-B is capable of interacting with Hsp40 and sAnkl is capable of interacting with obscurin and titin, but the function of C-terminal domain of ankyrin-G remains unknown. To identify proteins interacting C-terminus of ankyrin-G, we used the C-terminus of ankyrin-G as a bait for a yeast two-hybrid screen of brain cDNA library. Approximately 1.33$\times$l0$^6$ transformants were screened, of which 13 positive clones were obtained as determined by activation of HIS3, ADE2 and MELl reporter genes. Sequence analyses of these 13 plasmids revealed that cDNA inserts of 13 colonies showed highly homologous to 11 genes, including 5 known (i.e., Na$^+$/K$^+$ ATPase $\beta$1, SERBPl, UTF2, cytochrome C oxidase and collagen IV $\alpha$2) and 6 unknown genes. The evaluation of the proteins that emerge from these experiments provides a rational approach to investigate the those proteins significant in interaction with ankyrin-G.

• 한국독성학회:학술대회논문집
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• 한국독성학회 2006년도 추계학술대회
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• pp.46-54
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• 2006

The methylmercury (MeHg) is a toxic environmental pollutant, causing serious neurological and developmental effects in humans. Recent epidemiological studies have indicated that ingestion of MeHg in fish during pregnancy can result in neuroethological effects in the offspring. However, the mechanism underlying the MeHg-toxicity is not fully understood. To elucidate the mechanisms of toxicity of MeHg and of defense against MeHg, we searched for factors that determine the sensitivity of yeast cells to MeHg, and found that overexpression of Cdc34, a ubiquitin-conjugating enzyme (E2) that is a component of the ubiquitin-proteasome (UP) system, induces a resistance to MeHg toxicity in both yeast and human cells. The UP system is involved in the intracellular degradation of proteins. When Cdc34 is overexpressed in cells, ubiquitination reactions are activated and the degradation of certain proteins by the UP system is enhanced. Therefore, it seems likely that certain as-yet-unidentified proteins that increase MeHg toxicity might exist in cons and that toxicity might be reduced by the enhanced degradation of such proteins, mediated by the UP system, when Cdc34 is overexpressed. SCF ubiquitin-ligase is a component of UP system and consists of Skpl, the scaffold protein Cdc53, the RING-finger protein Hrt1, and one member of the family of F-box proteins. The F-box proteins directly bind to the substrates and are the determinants of substrate specificity of SCF. Therefore, we searched for the f-box protein that cofers resistance to MeHg, and found that overexpression of Hrt3 or Yi1224w induced resistance to MeHg toxicity in yeast cells. Since the protein(5) that enhance toxicity of MeHg might plausibly be induced in substrates of both f-box proteins, we next searched for substrate proteins that are recognized by Hrt3 or Y1r224w using two-hybrid screen. We found that Did3 or Crsl interacts with Hrt3; and Eno2 interacts with Yir224w. The yeast cells that overexpressed each those proteins showed hypersensitivity to MeHg, respectively, indicating that those proteins enhance the MeHg toxicity. Both Dld3 and Eno2 are proteins involved in the synthesis of pyruvate, and overexpression of both proteins might induce increase in interacellular levels of pyruvate. Deletion of Yi1006w that transports pyruvate into the mitochondria induced aresistance to MeHg. These results suggest that the promotion of the pyruvate irdlowinto the mitochondria might enhance MeHg toxicity. This study providesimportant keyfor the elucidauon of the molecular mechanism of MeHg toxicity.