Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of Glyoxysomes in etiolated cotyledons of Arabidopsis thaliana
Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of Glyoxysomes in etiolated cotyledons of Arabidopsis thaliana
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DOI：
10.1093/pcp/pcg145
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发表时间：
2003-10-01
影响因子：
4.9
通讯作者：
Nishimura, M
中科院分区：
文献类型：
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作者：
Fukao, Y;Hayashi, M;Nishimura, M
乙醛酸酶体存在于黄化的子叶中，并含有用于植物异生的酶，这构成了乙醛酸酶体的主要功能。然而，在拟南芥基因组中存在281个似乎与过氧化物酶体功能相关的基因，这意味着许多未知的蛋白质存在于乙醛酸酶体中。为了更好地了解乙醛酸酶体的功能，我们对黄化的拟南芥子叶进行了乙醛酸酶体蛋白质组学分析。其中19个蛋白质被鉴定为乙醛酸酶体蛋白，包括13个新蛋白质，其中之一是乙醛酸酶体蛋白激酶I（GPK 1）。我们用RT-PCR方法克隆了GPK 1的cDNA，并对其进行了鉴定。从GPK 1 cDNA推导的氨基酸序列具有疏水区，推定的蛋白激酶结构域，和可能的PTS 1基序。使用Percoll密度梯度上收集的级分进行免疫印迹分析证实GPK 1定位于乙醛酸体中。亚细胞器定位和蛋白酶敏感性的分析表明，GPK 1是本地化的glyoxysomal膜作为外周膜蛋白和推定的激酶结构域位于glyoxysomes内。Glyoxysomal蛋白在各种金属离子和[γ-P-32]ATP的存在下被很好地磷酸化，其中一种通过免疫沉淀被鉴定为硫解酶。免疫抑制磷酸化的glyoxysomes表明，GPK 1磷酸化的40 kDa的蛋白质。这些结果表明蛋白磷酸化系统在乙醛酸酶体中起作用。
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.