Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis
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Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis. / Parsons, Harriet T; Christiansen, Katy; Knierim, Bernhard; Carroll, Andrew; Ito, Jun; Batth, Tanveer S; Smith-Moritz, Andreia M; Morrison, Stephanie; McInerney, Peter; Hadi, Masood Z; Auer, Manfred; Mukhopadhyay, Aindrila; Petzold, Christopher J; Scheller, Henrik V; Loqué, Dominique; Heazlewood, Joshua L.
In: Plant Physiology, Vol. 159, No. 1, 05.2012, p. 12-26.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis
AU - Parsons, Harriet T
AU - Christiansen, Katy
AU - Knierim, Bernhard
AU - Carroll, Andrew
AU - Ito, Jun
AU - Batth, Tanveer S
AU - Smith-Moritz, Andreia M
AU - Morrison, Stephanie
AU - McInerney, Peter
AU - Hadi, Masood Z
AU - Auer, Manfred
AU - Mukhopadhyay, Aindrila
AU - Petzold, Christopher J
AU - Scheller, Henrik V
AU - Loqué, Dominique
AU - Heazlewood, Joshua L
PY - 2012/5
Y1 - 2012/5
N2 - 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.
AB - 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.
KW - Apyrase
KW - Arabidopsis
KW - Arabidopsis Proteins
KW - Base Sequence
KW - Cell Wall
KW - Centrifugation, Density Gradient
KW - Chromatography, Liquid
KW - Enzyme Assays
KW - Genes, Plant
KW - Genetic Complementation Test
KW - Glycosylation
KW - Golgi Apparatus
KW - Immunoblotting
KW - Intracellular Membranes
KW - Microscopy, Electron, Transmission
KW - Molecular Sequence Data
KW - Plant Cells
KW - Proteome
KW - Proteomics
KW - Pyrophosphatases
KW - Saccharomyces cerevisiae
U2 - 10.1104/pp.111.193151
DO - 10.1104/pp.111.193151
M3 - Journal article
C2 - 22430844
VL - 159
SP - 12
EP - 26
JO - Plant Physiology
JF - Plant Physiology
SN - 0032-0889
IS - 1
ER -
ID: 68160606