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Plant and Cell Physiology Advance Access originally published online on February 15, 2008
Plant and Cell Physiology 2008 49(4):526-539; doi:10.1093/pcp/pcn027
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Proteomic Analysis of Highly Purified Peroxisomes from Etiolated Soybean Cotyledons

Yuko Arai, Makoto Hayashi and Mikio Nishimura*

Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444-8585 Japan

*Corresponding author: E-mail, mikosome{at}nibb.ac.jp; Fax, +81-564-55-7505.


  Abstract

To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid β-oxidation, the glyoxylate cycle, photorespiratory glycolate metabolism, stress response and metabolite transport. We also show experimental evidence that plant peroxisomes contain a short-chain dehydrogenase/reductase family protein, enoyl-CoA hydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-like protein and a voltage-dependent anion-selective channel protein.

Keywords: Expressed sequence tag - Glyoxysome - Matrix-assisted laser desorption ionization time-of-flight mass spectrometry - Peroxisome - Proteomics - Soybean

Abbreviations: CaMV, cauliflower mosaic virus; 2-D, 2-dimensional; ER, endoplasmic reticulum; EST, expressed sequence tag; GFP, green fluorescent protein; HRP, horseradish peroxidase; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; mRFP, monomeric red fluorescent protein; PIR, protein information resource; PMF, peptide mass fingerprinting; PTS, peroxisomal targeting signal; RT–PCR, reverse transcription–PCR; SDR, short-chain dehydrogenase/reductase; TC, tentative consensus.

(Received January 16, 2008; Accepted February 12, 2008)
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