title: Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis.
abstract: The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.
authors.affiliation: Joint BioEnergy Institute and Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
authors.forename: Harriet T
authors.initials: HT
authors.lastname: Parsons
authors.forename: Katy
authors.initials: K
authors.lastname: Christiansen
authors.forename: Bernhard
authors.initials: B
authors.lastname: Knierim
authors.forename: Andrew
authors.initials: A
authors.lastname: Carroll
authors.forename: Jun
authors.initials: J
authors.lastname: Ito
authors.forename: Tanveer S
authors.initials: TS
authors.lastname: Batth
authors.forename: Andreia M
authors.initials: AM
authors.lastname: Smith-Moritz
authors.forename: Stephanie
authors.initials: S
authors.lastname: Morrison
authors.forename: Peter
authors.initials: P
authors.lastname: McInerney
authors.forename: Masood Z
authors.initials: MZ
authors.lastname: Hadi
authors.forename: Manfred
authors.initials: M
authors.lastname: Auer
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.forename: Henrik V
authors.initials: HV
authors.lastname: Scheller
authors.forename: Dominique
authors.initials: D
authors.lastname: Loqué
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Heazlewood
mesh_terms: D001081:Apyrase / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002473:Cell Wall / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D057075:Enzyme Assays; D017343:Genes, Plant; D005816:Genetic Complementation Test; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D059828:Plant Cells / Q000201:enzymology / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D020543:Proteome; D011755:Pyrophosphatases; C010357:guanosine-diphosphatase; C522733:APY1 protein, Arabidopsis; D001081:Apyrase