Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS.
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.
Phee
Bong-Kwan
BK
Graduate School of Biotechnology and Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea.
Shin
Dong Ho
DH
Cho
Jin-Hwan
JH
Kim
Seong-Hee
SH
Kim
Jeong-Il
JI
Lee
Youn-Hyung
YH
Jeon
Jong-Seong
JS
Bhoo
Seong Hee
SH
Hahn
Tae-Ryong
TR
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
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D010834:Phytochrome