Journal Article

Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of <i>Arabidopsis thaliana</i>

Yoichiro Fukao, Makoto Hayashi, Ikuko Hara-Nishimura and Mikio Nishimura

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 44, issue 10, pages 1002-1012
Published in print October 2003 | ISSN: 0032-0781
Published online October 2003 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcg145
Novel Glyoxysomal Protein Kinase, GPK1, Identified by Proteomic Analysis of Glyoxysomes in Etiolated Cotyledons of Arabidopsis thaliana

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  • Biochemistry
  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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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 1 (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 [g-32P]ATP, and one of them is identified as thiolase by immunoprecipitation. Immuno-inhibition of phosphorylation in glyoxysomes suggested that GPK1 phosphorylates a 40-kDa protein. These results show that protein phosphorylation systems are operating in glyoxysomes.

Keywords: Keywords: Arabidopsis — Etiolated cotyledon — Glyoxysomes — Protein kinase — Protein phosphorylation — Proteome.; Abbreviations: CAO, copper amine oxidase; CAT, catalase; COX, cytochrome c oxidase; CS, citrate synthase; 2D, two-dimensional; GPK1, glyoxysomal protein kinase 1; ICL, isocitrate lyase; IDH, isocitrate dehydrogenase; IPG, immobilized pH gradient; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MDH, malate dehydrogenase; pAPX, peroxisomal ascorbate peroxidase; PMP, peroxisomal membrane protein; PTS, peroxisomal targeting signal.

Journal Article.  7820 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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