RESEARCH ARTICLE Differential proteomic analysis of the endoplasmic reticulum from developing and germinating seeds of castor (Ricinus communis) identifies seed protein precursors as significant components of the endoplasmic reticulum Daniel J. Maltman, Stephen M. Gadd, William J. Simon and Antoni R. Slabas School of Biological and Biomedical Sciences, Durham University, Durham, UK The endoplasmic reticulum is a major compartment of storage protein and lipid biosynthesis. Maximal synthesis of these storage compounds occurs during seed development with breakdown occurring during germination. In this study, we have isolated four independent preparations of ER from both developing and germinating seeds of castor bean (Ricinus communis) and used 2-D DIGE, and a combination of PMF and MS/MS sequencing, to quantify and identify differences in protein complement at both stages. Ninety protein spots in the developing seeds are up-regulated and 19 individual proteins were identified, the majority of these are intermediates of seed storage synthesis and protein folding. The detection of these transitory storage proteins in the ER is dis- cussed in terms of protein trafficking and processing. In germinating seed ER 15 spots are ele- vated, 5 of which were identified, amongst them was malate synthetase which is a component of the glyoxysome which is believed to originate from the ER. Notably no proteins involved in complex lipid biosynthesis were identified in the urea soluble ER fraction indicating that they are probably all integral membrane proteins. Received: September 14, 2006 Revised: January 15, 2007 Accepted: February 6, 2007 Keywords: Castor / Endoplasmic reticulum / Storage proteins Proteomics 2007, 7, 1513–1528 1513 1 Introduction The ER is part of the endomembrane system of plants. It serves a number of diverse functions and represents a tran- sitory vehicle through which storage proteins pass during their biosynthesis as well as proteins destined for the extra- cellular matrix of plants. It thus plays an important role in the early events of the secretory pathway of protein synthesis which includes docking of proteins to the ER by signal recognition particles, protein folding and glycosylation as well as the formation of disulphide bridges between proteins [1]. In yeast and animals, as part of the unfolded protein re- sponse, the ER has been shown to be part of a stress signal- ling pathway of gene regulation [2]. In plants much of our understanding of the sequence of events in secretory protein synthesis has been derived from radiolabelling experiments combined with a firm understanding of the primary structure of the mature proteins derived from protein sequencing Correspondence: Professor Antoni R. Slabas, School of Biologi- cal and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK E-mail: a.r.slabas@durham.ac.uk Fax: 144-191–3341295 Abbreviations: PDI, protein disulphide-isomerase; RCA, Ricinus communis agglutinin; TAG, triacylglycerol DOI 10.1002/pmic.200600694  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com