Aproteomicanalysisoforganellesfrom Arabidopsis thaliana We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homoge- neous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endo- plasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electro- phoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define spe- cific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisa- tion of novel plant proteins. Keywords: Arabidopsis thaliana / Proteomics / Organelle proteins EL 4123 TracyA.Prime 1 D.JanineSherrier 1* PiersMahon 1 LenC.Packman 2 PaulDupree 1 1 Plant Cell Biology Laboratory 2 Protein and Nucleic Acid Facility, Department of Biochemistry, Cambridge, UK 1 Introduction The unique biosynthetic and catabolic processes of plant cells are highly compartmentalised. The synthesis of amino and fatty acids in plastids, extracellular matrix poly- saccharides in the Golgi apparatus and lipids in the endo- plasmic reticulum (ER) are separated from each other and from the catabolic activities of mitochondria. Further- more, protein storage and degradation functions can be separated into two or more vacuoles within a single cell [1, 2]. Each compartment is bound by a membrane that contains and encloses a unique complement of proteins. Knowledge of the protein composition is still relatively rudimentary despite being essential in order to under- stand the biogenesis and function of these plant organ- elles. An important approach to understanding plant cell biology has been through the use of mutants, allowing single gene mutations to be associated with a specific pheno- type. This has proved to be especially powerful in the model plant, Arabidopsis thaliana [3]. A reverse genetics approach can now also be used, and several populations of insertional mutagenised plants are currently available (see e.g. [4]). However, despite the power of these ap- proaches, results can be obscured by genetic redun- dancy. There is increasing evidence that plant genomes, including Arabidopsis, contain many gene duplications [5, 6]. A further invaluable approach has been to purify enzymes or proteins on the basis of their activity; this, however, requires the development of a specific biochem- ical assay. With the advent of genome sequence informa- tion, reproducible 2-D SDS-PAGE, and sensitive mass spectrometric protein analysis techniques, proteomics provides an alternative approach to identify many proteins in parallel. By linking protein identity with subcellular local- isation, putative functions can be assigned to novel pro- teins predicted in genome sequencing projects. Plant proteome projects based on 2-D SDS-PAGE have recently been initiated in a few laboratories [7]. Most have investigated protein composition of seeds or tissues, or have analysed protein expression changes in mutants or during stress [7, 8]. There have been few projects that use 2-D SDS-PAGE to investigate organelle composition. The work in early papers demonstrated different polypep- tide compositions of membrane fractions [9±12]. Re- cently, Peltier and co-workers [13] identified thylakoid peripheral and luminal proteins from pea chloroplasts, and 17 putative peribacteroid membrane proteins have been identified in soybean [14]. One of the first organelle proteome projects in plants was the European Commis- sion framework IV project to characterise Arabidopsis plasma membrane (PM) proteins [15], and the work de- scribed here contributes to that project. Correspondence: Dr. Paul Dupree, Plant Cell Biology Laborato- ry, Department of Biochemistry, Building O, Downing Site, Cambridge, CB2 IQW, UK E-mail: p.dupree@bioc.cam.ac.uk Fax: +44-1223-333345 Abbreviations: CIM, callus induction medium; EM, electron mi- croscopy; ER, endoplasmic reticulum; PDI, protein disulphide isomerase; PM, plasma membrane 3488 Electrophoresis 2000, 21, 3488±3499  WILEY-VCH Verlag GmbH, 69451 Weinheim, 2000 0173-0835/00/1616-3488 $17.50+.50/0 * Current address: Department of Plant and Soil Sciences and Delaware Biotechnology Institute, University of Delaware, Newark, DE 19717, USA