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Characterization of microtubule-binding and dimerizatino activity of Giardia lamblia end-binding 1 protein

 Juri Kim  ;  Sara Nagami  ;  Kyu-Ho Lee  ;  Soon-Jung Park 
 PLOS ONE, Vol.9(5) : e97850, 2014 
Journal Title
Issue Date
Cell Cycle Proteins/genetics ; Cell Cycle Proteins/metabolism ; Cell Nucleus/metabolism ; Cell Nucleus/ultrastructure ; Gene Expression Regulation ; Genetic Complementation Test ; Giardia lamblia/genetics ; Giardia lamblia/metabolism* ; Giardia lamblia/ultrastructure ; Hemagglutinins/chemistry ; Hemagglutinins/genetics ; Hemagglutinins/metabolism ; Microtubule Proteins/genetics ; Microtubule Proteins/metabolism ; Microtubule-Associated Proteins/antagonists & inhibitors ; Microtubule-Associated Proteins/chemistry ; Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/metabolism* ; Microtubules/metabolism* ; Microtubules/ultrastructure ; Mitosis ; Morpholinos/genetics ; Morpholinos/metabolism ; Mutation ; Protein Binding ; Protein Multimerization ; Protozoan Proteins/antagonists & inhibitors ; Protozoan Proteins/chemistry ; Protozoan Proteins/genetics ; Protozoan Proteins/metabolism* ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism* ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/genetics ; Saccharomyces cerevisiae Proteins/metabolism ; Trophozoites/metabolism ; Trophozoites/ultrastructure
End-binding 1 (EB1) proteins are evolutionarily conserved components of microtubule (MT) plus-end tracking protein that regulate MT dynamics. Giardia lamblia, with two nuclei and cytoskeletal structures, requires accurate MT distribution for division. In this study, we show that a single EB1 homolog gene of G. lamblia regulates MT dynamics in mitosis. The haemagglutinin-tagged G. lamblia EB1 (GlEB1) localizes to the nuclear envelopes and median bodies, and is transiently present in mitotic spindles of dividing cells. Knockdown of GlEB1 expression using the morpholinos-based anti-EB1 oligonucleotides, resulted in a significant defect in mitosis of Giardia trophozoites. The MT-binding assays using recombinant GlEB1 (rGlEB1) proteins demonstrated that rGlEB1102-238, but not rGlEB11-184, maintains an MT-binding ability comparable with that of the full length protein, rGlEB11-238. Size exclusion chromatography showed that rGlEB1 is present as a dimer formed by its C-terminal domain and a disulfide bond. In vitro-mutagenesis of GlEB1 indicated that an intermolecular disulfide bond is made between cysteine #13 of the two monomers. Complementation assay using the BIM1 knockout mutant yeast, the yeast homolog of mammalian EB1, indicated that expression of the C13S mutant GlEB1 protein cannot rescue the mitotic defect of the BIM1 mutant yeast. These results suggest that dimerization of GlEB1 via the 13th cysteine residues plays a role during mitosis in Giardia.
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1. College of Medicine (의과대학) > Dept. of Tropica Medicine (열대의학교실) > 1. Journal Papers
Yonsei Authors
Kim, Ju Ri(김주리) ORCID logo https://orcid.org/0000-0001-8270-7584
Park, Soon Jung(박순정) ORCID logo https://orcid.org/0000-0002-0423-1944
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