Characterization of microtubule-binding and dimerization activity of Giardia lamblia end-binding 1 protein.
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.
Department of Environmental Medical Biology and Institute of Tropical Medicine, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Juri
J
Kim
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea.
Sara
S
Nagami
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, South Korea.
Kyu-Ho
KH
Lee
Department of Environmental Medical Biology and Institute of Tropical Medicine, Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Soon-Jung
SJ
Park
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D016829:Giardia lamblia / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008938:Mitosis; D060172:Morpholinos / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D053058:Trophozoites / Q000378:metabolism / Q000648:ultrastructure
C119683:BIM1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D006388:Hemagglutinins; D008868:Microtubule Proteins; D008869:Microtubule-Associated Proteins; D060172:Morpholinos; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins