title: Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS.
abstract: Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.
authors.affiliation: Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea.
authors.forename: Bong-Kwan
authors.initials: BK
authors.lastname: Phee
authors.forename: Dong Ho
authors.initials: DH
authors.lastname: Shin
authors.forename: Jin-Hwan
authors.initials: JH
authors.lastname: Cho
authors.forename: Seong-Hee
authors.initials: SH
authors.lastname: Kim
authors.forename: Jeong-Il
authors.initials: JI
authors.lastname: Kim
authors.forename: Youn-Hyung
authors.initials: YH
authors.lastname: Lee
authors.forename: Jong-Seong
authors.initials: JS
authors.lastname: Jeon
authors.forename: Seong Hee
authors.initials: SH
authors.lastname: Bhoo
authors.forename: Tae-Ryong
authors.initials: TR
authors.lastname: Hahn
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018744:DNA, Plant / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D047468:Immunoprecipitation; D008954:Models, Biological; D016254:Mutagenesis, Insertional; D010834:Phytochrome / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D010834:Phytochrome