Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel components involved in plant cell wall biosynthesis

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

SN - 0032-0889

VL - 159

SP - 12

EP - 26

JO - Plant Physiology

JF - Plant Physiology

IS - 1

ER -