Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of Glyoxysomes in etiolated cotyledons of Arabidopsis thaliana

被引:60
作者
Fukao, Y
Hayashi, M
Hara-Nishimura, I
Nishimura, M [1 ]
机构
[1] Natl Inst Basic Biol, Dept Cell Biol, Okazaki, Aichi 4448585, Japan
[2] Grad Univ Adv Studies, Sch Life Sci, Dept Mol Biomech, Okazaki, Aichi 4448585, Japan
[3] Kyoto Univ, Grad Sch Sci, Dept Bot, Sakyo Ku, Kyoto 6068502, Japan
基金
日本学术振兴会; 日本科学技术振兴机构;
关键词
Arabidopsis; etiolated cotyledon; glyoxysomes; protein kinase; protein phosphorylation; proteome;
D O I
10.1093/pcp/pcg145
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Glyoxysomes are present in etiolated cotyledons and contain enzymes for gluconeogenesis, which constitutes the major function of glyoxysomes. However, 281 genes seemingly related to peroxisomal functions occur in the Arabidopsis genome, implying that many unidentified proteins are present in glyoxysomes. To better understand the functions of glyoxysomes, we performed glyoxysomal proteomic analysis of etiolated Arabidopsis cotyledons. Nineteen proteins were identified as glyoxysomal proteins, including 13 novel proteins, one of which is glyoxysomal protein kinase I (GPK1). We cloned GPK1 cDNA by RT-PCR and characterized GPK1. The amino acid sequence deduced from GPK1 cDNA has a hydrophobic region, a putative protein kinase domain, and a possible PTS1 motif. Immunoblot analysis using fractions collected on a Percoll density gradient confirmed that GPK1 is localized in glyoxysomes. Analysis of suborganellar localization and protease sensitivity showed that GPK1 is localized on glyoxysomal membranes as a peripheral membrane protein and that the putative kinase domain is located inside the glyoxysomes. Glyoxysomal proteins are phosphorylated well in the presence of various metal ions and [gamma-P-32]ATP, and one of them is identified as thiolase by immunoprecipitation. Immunoinhibition of phosphorylation in glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in glyoxysomes.
引用
收藏
页码:1002 / 1012
页数:11
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