Abstract
Vacuoles play central roles in plant growth, development, and stress responses. To better understand vacuole function and biogenesis we have characterized the vegetative vacuolar proteome from Arabidopsis thaliana. Vacuoles were isolated from protoplasts derived from rosette leaf tissue. Total purified vacuolar proteins were then subjected either to multidimensional liquid chromatography/tandem mass spectrometry or to one-dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry (nano-LC MS/MS). To ensure maximum coverage of the proteome, a tonoplast-enriched fraction was also analyzed separately by one-dimensional SDS-PAGE followed by nano-LC MS/MS. Cumulatively, 402 proteins were identified. The sensitivity of our analyses is indicated by the high coverage of membrane proteins. Eleven of the twelve known vacuolar-ATPase subunits were identified. Here, we present evidence of four tonoplast-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), representing each of the four groups of SNARE proteins necessary for membrane fusion. In addition, potential cargo of the N- and C-terminal propeptide sorting pathways, association of the vacuole with the cytoskeleton, and the vacuolar localization of 89 proteins of unknown function are identified. A detailed analysis of these proteins and their roles in vacuole function and biogenesis is presented.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Arabidopsis / chemistry
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Arabidopsis / metabolism*
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Arabidopsis Proteins / chemistry
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Arabidopsis Proteins / isolation & purification
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Arabidopsis Proteins / metabolism*
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Cytoskeleton / chemistry
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Cytoskeleton / metabolism
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Membrane Fusion / physiology
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Membrane Proteins / chemistry
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism*
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Molecular Sequence Data
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Peptides / metabolism
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Plant Leaves / chemistry
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Plant Leaves / metabolism
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Protein Structure, Tertiary / physiology
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Protein Subunits / chemistry
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism
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Protein Transport / physiology
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Proteome / chemistry
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Proteome / isolation & purification
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Proteome / metabolism*
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Proteomics / methods
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Protoplasts / chemistry
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Protoplasts / metabolism
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SNARE Proteins
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Signal Transduction / physiology
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Vacuolar Proton-Translocating ATPases / chemistry
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Vacuolar Proton-Translocating ATPases / isolation & purification
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Vacuolar Proton-Translocating ATPases / metabolism
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Vacuoles / chemistry
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Vacuoles / metabolism*
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Vesicular Transport Proteins / chemistry
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Vesicular Transport Proteins / isolation & purification
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Vesicular Transport Proteins / metabolism
Substances
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Arabidopsis Proteins
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Membrane Proteins
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Peptides
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Protein Subunits
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Proteome
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SNARE Proteins
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Vesicular Transport Proteins
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Vacuolar Proton-Translocating ATPases