• Title/Summary/Keyword: Yeast two-hybrid screening

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Genetic Screening of the Dazl-Interacting Protein Genes

  • Lee, Kyung-Ho;Lee, Seong-Ju;Rhee, Kun-Soo
    • Animal cells and systems
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    • v.10 no.4
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    • pp.227-231
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    • 2006
  • Micro-deletions at specific loci of the Y chromosome have been observed frequently in male infertility patients, suggesting that genes in these regions are involved in male germ cell development. DAZ is a representative male infertility gene at the AZFc locus of the Y chromosome. Since DAZ contains an RNA binding motif along with so-called a DAZ domain, it was proposed to participate in RNA metabolism during spermatogenesis. A mouse gene homologous to the human DAZ gene has been cloned and named Dazl (DAZlike). Dazl is autosomal and expressed in the testis and also at a low level in the ovary. Male mice homozygous for the Dazl null allele have small testes with a few spermatogonia and almost complete absence of germ cells beyond the spermatogonial stage, suggesting the requirement of Dazl for entry or progression through meiosis. However, its exact cellular functions have not been understood yet. In order to investigate cellular functions of Dazl, we decided to isolate candidate interacting protein genes of the mouse Dazl, using yeast two-hybrid screening. A number of candidate Dazlinteracting proteins have been isolated, such as Bprp, Acf, Hgs, Murr1, Nbak3 and Ranbp9, but dynein light chain 1 (Dlc1) was most predominant. A strong interaction of Dazl with Dlc1 suggests that Dazl might function as an mRNA adaptor to the dynein motor complex.

The β Subunit of Heterotrimeric G Protein Interacts Directly with Kinesin Heavy Chains, Kinesin-I (Kinesin-I의 kinesin heavy chains과 직접 결합하는 heterotrimeric G protein의 β subunit의 규명)

  • Seog, Dae-Hyun
    • Journal of Life Science
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    • v.20 no.8
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    • pp.1166-1172
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    • 2010
  • Kinesin-I exists as a tetramer of two heavy chains (KHCs, also called KIF5s), which contain the amino (N)-terminal motor domain and carboxyl (C)-terminal domain, as well as two light chains (KLCs), which bind to the KIF5s (KIF5A, KIF5B and KIF5C) stalk region. To identify the interaction proteins for KIF5A, yeast two-hybrid screening was performed and a specific interaction with the ${\beta}$ subunit of heterotrimeric G proteins ($G{\beta}$) was found. $G{\beta}$ bound to the amino acid residues between 808 and 935 of KIF5A and to other KIF5 members in the yeast two-hybrid assay. The WD40 repeat motif of $G{\beta}$ was essential for interaction with KIF5A. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to KIF5s specifically co-immunoprecipitated KIF5s associated with heterotrimeric G proteins from mouse brain extracts. These results suggest that kinesin-I motor protein transports heteroterimeric G protein attachment vesicles along microtubules in the cell.

Interaction between IgE-Dependent Histamine-Releasing Factor and Triosephosphate Isomerase in HeLa Cells (HeLa 세포에서 IgE-dependent Histamine-Releasing Factor와 Triosephosphate Isomerase의 상호작용 규명)

  • Moon Ji-Ae;Kim Hwa-Jung;Lee Kyunglim
    • Microbiology and Biotechnology Letters
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    • v.33 no.4
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    • pp.255-259
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    • 2005
  • IgE-dependent histamine-releasing factor (HRF) is found extracellularly to regulate the degranulation process of histamine in mast cells and basophils and known to play a predominant role in the pathogenesis of chronic allergic disease. HRF has been also identified in the intracellular region of the cell. Previously, we reported that HRF interacts with the 3rd cytoplasmic domain of the alpha subunit of Na,K-ATPase. To understand the molecular mechanism of the regulation of Na, K-ATPase activity by HRF, we investigated the interaction between HRF and TPI since TPI was obtained as HRF-interacting protein in HeLa cDNA library, using yeast two hybrid screening. Domain mapping study of the interaction between HRF and TPI revealed that the C-terminal region of the residue 156-249 of TPI is involved in the interaction with HRF. The interaction between HRF and TPI was confirmed by immunoprecipitation from HeLa cell extracts. Our results suggest that TPI is a HRF-binding protein and the interaction between HRF and TPI nay thus affect Na, K-ATPase activity.

Archvillin C-Terminus-Binding Proteins in Human Skeletal Muscle

  • Chang, Goo-Rak
    • Biomedical Science Letters
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    • v.16 no.4
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    • pp.207-212
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    • 2010
  • Archivillin, a muscle-specific isoform of supervillin, is a component of the costameric cytoskeleton of muscle cells. The purpose of this study was to determine which protein in the skeletal muscle collaborates with archvillin C-terminus. For this purpose, a yeast two-hybrid screening of human skeletal muscle cDNA library was performed using the C-terminal region of archvillin as bait. This study shows that seven human skeletal muscle proteins, namely, nebulin, xeplin, archvillin, GAPDH, TOX4, PITRM1, and YME1L1 interact with archvillin C-terminus. Especially, xeplin is a newly discovered protein interacts with archvillin C-terminus. These results indicate that archvillin C-terminus acts as a bridge between nebulin and xeplin at costameres. Archvillin C-terminal region interacts with nebulin C-terminal region at Z-discs and interacts with xeplin at the vicinity of sarcolemma. I propose that these interactions may contribute to formation of costameric structure and muscle contraction.

The Identification of Proteins Interacting with CD1d (CD1d와 상호작용하는 단백질의 동정)

  • Hwang, Kwang-Woo;Chun, Tae-Hoon
    • YAKHAK HOEJI
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    • v.50 no.4
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    • pp.263-267
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    • 2006
  • CD1d is an unique antigen presenting molecule which provides antigenic repertoires to NKT cells. To examine molecules required for CD1d antigen presentation, we determined an interaction between CD1d and several endoplasmic reticulum (ER) resident molecular chaperones by co-immunoprecipitation. Results indicated that calnexin and calreticulin seem to be bound to mouse CD1d, but TAP and tapasin do not bind. Further, we screened an yeat two hybrid system to identify proteins that help mouse CD1d transportation in the cytosol. We found that two proteins, heat shock protein a sub-unit $(Hsp90{\alpha})$ and protein kinase C and casein kinase substrate in neurons 3 (PACSIN-3), interact with CD1d. Future study will be focus on the role of these molecules during the CD1d antigen presentation.

Direct Interaction of KIF5s and Actin-Based Transport Motor, Myo9s (KIF5s와 직접 결합하는 액틴 결합 운동단백질 Myo9s의 규명)

  • Seog, Dae-Hyun
    • 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.

Identification of the Interaction between Rat Translationally Controlled Tumor Protein/IgE-dependent Histamine Releasing Factor and Myosin Light Chain

  • Kim, Min-Jeong;Jung, Jae-Hoon;Choi, Eung-Chil;Park, Hae-Young;Lee, Kyung-Lim
    • BMB Reports
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    • v.34 no.6
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    • pp.526-530
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    • 2001
  • The translationally controlled tumor protein (TCTP), also known as the IgE-dependent histamine releasing factor (HRF), was used in the yeast two-hybrid system to screen the interacting molecules. We obtained the N-terminus truncated rat fast myosin alkai light chain from the rat skeletal muscle cDNA library in the screening. Since either TCTP/HRF or the myosin light chain is known to be associated with histamine secretion from RBL-2H3 cells, we investigated the possible interaction between rat TCTP/HRF and nonmuscle myosin light chain in these cells. We used affinity chromatography and coimmunoprecipitation. Our data suggests that HRF and the myosin light chain interact, which may play an important role in histamine release in RBL-2H3 cells.

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Identification of a Cellular Protein Interacting with RNA Polymerase of Hepatitis C Virus

  • Park, Kyu-Jin;Choi, Soo-Ho;Koh, Moon-Soo;Kim, Sung-Wan;Hwang, Soon-Bong
    • BMB Reports
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    • v.33 no.1
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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.

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Screening of Domain-specific Target Proteins of Polo-like Kinase 1: Construction and Application of Centrosome/Kinetochore-specific Targeting Peptide

  • Ji, Jae-Hoon;Jang, Young-Joo
    • BMB Reports
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    • v.39 no.6
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    • pp.709-716
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    • 2006
  • Mammalian polo-like kinase 1 (Plk1) acts at various stages in early and late mitosis. Plk1 localizes at the centrosome and maintains this position through mitosis. Thereafter Plk1 moves to the kinetochore and midbody region, important sites during chromosome separation and cytokinesis. The catalytic domain of Plk1 is in the N-terminus region, whereas the non-catalytic region in the C-terminus of Plk1 has a conserved motif, named the Polobox. This motif is critical for Plk localization. EGFP proteins fused with the N-terminus and C-terminus of Plk1 localize in the nucleus and centrosomes, respectively. The core sequences of the polo-box (50 amino acids) also localize in Plk1 target organelles. To screen for domain-specific target proteins of Plk1, we constructed an N-terminal domain and a tandem repeat polo-box motif, and used them as templates in a yeast two-hybrid screen. The HeLa cell cDNA library indicated several proteins including the centrosome/kinetochore components or regulators, to be characterized as positive clones. Through in vitro protein binding analyses, we confirmed an interaction between these proteins and Plk1. The data reported from this study indicate that the N- and C- termini of Plk1 may function through recruitment and/or activation of domain-specific target proteins in dividing cells. Additionally, tandem repeats of the conserved core motif of the polo-box are sufficient for targeting and may be useful as a centrosome/kinetochore-specific targeting peptide.

Characterization of ERp29 and ADP-Ribosylation Factor 5 Interaction (ERp29와 ADP-ribosylation factor 5의 결합특성)

  • Kwon, Ki-Sang;Seog, Dae-Hyun;Kim, Seung-Whan;Yu, Kweon;Kwon, O-Yu
    • Journal of Life Science
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    • v.21 no.4
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    • pp.613-615
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    • 2011
  • ERp29 is a endoplasmic reticulum (ER) lumenal resident protein that shows sequence similarity to the protein disulfide isomerase family. Its biological function is thought to play a role in the processing of secretory proteins within the ER, possibly by participating in the folding of proteins in the ER. Although some data on ERp29 have been reported, its normal functions are still unclear. To gain insights into the function of ERp29, we identified ARF5 protein as a protein that interacts with ERp29 using yeast two-hybrid screening and GST pull-down assay. Interaction between ERp29 and ARF5 was detected under normal cell conditions but not under ER stress conditions. This result may provide a clue for understanding ERp29 biological functions.