title: Proteomic Characterization of Golgi Membranes Enriched from Arabidopsis Suspension Cell Cultures.
abstract: The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.
authors.affiliation: Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg C, Denmark.|Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94702, USA.
authors.forename: Sara Fasmer
authors.initials: SF
authors.lastname: Hansen
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC, 3010, Australia.
authors.forename: Berit
authors.initials: B
authors.lastname: Ebert
authors.affiliation: ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC, 3010, Australia.
authors.forename: Carsten
authors.initials: C
authors.lastname: Rautengarten
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94702, USA. jheazlewood@unimelb.edu.au.|ARC Centre of Excellence in Plant Cell Walls, School of BioSciences, The University of Melbourne, Parkville, VIC, 3010, Australia. jheazlewood@unimelb.edu.au.
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D059828:Plant Cells / Q000737:chemistry* / Q000378:metabolism
keywords: Density gradient centrifugation; Fluorescent protein; Golgi apparatus; Subcellular localization