title: Dissecting the subcellular compartmentation of proteins and metabolites in arabidopsis leaves using non-aqueous fractionation.
abstract: Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin-Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite-protein analysis on a single tissue sample.
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Arrivault
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Manuela
authors.initials: M
authors.lastname: Guenther
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Alexandra
authors.initials: A
authors.lastname: Florian
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Beatrice
authors.initials: B
authors.lastname: Encke
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Regina
authors.initials: R
authors.lastname: Feil
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany; §Stellenbosch University, Private Bag X1, Matieland 7602, Stellenbosch, South Africa;
authors.forename: Daniel
authors.initials: D
authors.lastname: Vosloh
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: John E
authors.initials: JE
authors.lastname: Lunn
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany; ¶National University of Ireland, University Rd., Galway, Ireland;
authors.forename: Ronan
authors.initials: R
authors.lastname: Sulpice
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Alisdair R
authors.initials: AR
authors.lastname: Fernie
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany;
authors.forename: Mark
authors.initials: M
authors.lastname: Stitt
authors.affiliation: From the ‡Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany; ‖Department of Plant Systems Biology, Universität Hohenheim, 70593 Stuttgart, Germany wschulze@uni-hohenheim.de.
authors.forename: Waltraud X
authors.initials: WX
authors.lastname: Schulze
mesh_terms: D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods*; D018515:Plant Leaves / Q000378:metabolism; D013347:Subcellular Fractions
chemical_list: D029681:Arabidopsis Proteins