Mapping the Arabidopsis organelle proteome - PubMed (original) (raw)

. 2006 Apr 25;103(17):6518-23.

doi: 10.1073/pnas.0506958103. Epub 2006 Apr 17.

Svenja Hester, Ian P Shadforth, John Runions, Thilo Weimar, Sally L Hanton, Julian L Griffin, Conrad Bessant, Federica Brandizzi, Chris Hawes, Rod B Watson, Paul Dupree, Kathryn S Lilley

Affiliations

Mapping the Arabidopsis organelle proteome

Tom P J Dunkley et al. Proc Natl Acad Sci U S A. 2006.

Abstract

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

PCA scores plot showing clustering of proteins according to their density gradient distributions and, therefore, localizations. iTRAQ reporter ion ratios were imported into

simca

10, logged, and preprocessed with unit-variance scaling before performing PCA analysis. Filled shapes indicate known organelle residents (marker proteins). Open shapes (or stars in the case of mitochondria/plastid) indicate proteins with predicted localizations that were confirmed based on their proximity to the corresponding marker proteins on the PCA scores plot. Small dots indicate proteins, without known or predicted localizations, that were assigned to an organelle by using PLS-DA, in conjunction with limited manual analysis. Small crosses indicate proteins that were not assigned to an organelle. Inverted triangles, vacuolar membrane; squares, ER; diamonds, PM; circles, known mitochondria/plastids; stars, predicted mitochondria/plastids; triangles, Golgi apparatus.

Fig. 2.

Fig. 2.

Fluorescent protein fusions to uncharacterized proteins to confirm LOPIT targeting predictions. (a_–_f) Golgi apparatus. Colocalization of uncharacterized protein with known Golgi apparatus markers is shown. (a) At1g04910-GFP marks small motile organelles. (b) ST-mRFP marks the Golgi apparatus. (c) Colocalization of the markers in a and b. (d) At1g31850 GMT1GFP marks small motile organelles. (e) ERD2-YFP marks Golgi apparatus strongly as bright spots and the ER weakly. (f) Colocalization of the markers in d and e. (Scale bars: 5 μm.) (g_–_i) ER localization. Colocalization of uncharacterized protein with known ER marker. (g) At3g44330-GFP marks a reticulate structure in the periphery of the cell. (h) YFP-HDEL marks the ER. (i) Colocalization of the markers in g and h. (Scale bars: 10 μm.) (j and k) Plasma membrane localization. (j) At1g14870-GFP. (Scale bar: 50 μm.) (k) Higher magnification of g. (Scale bars: 10 μm.) (l and m) Vacuolar membrane localization. (l) At2g47800-GFP. (Scale bar: 20 μm.) (m) Higher magnification of l. Note that membrane position toward the center of the cell relative to a chloroplast (arrowhead) confirms its identity as a vacuolar membrane. (Scale bar: 5 μm.)

Fig. 3.

Fig. 3.

PCA score plot showing positions of the ABC transporters, vacuolar sorting receptors, the unclassified V-ATPase V0a homolog, and GMT1 and GMT2 relative to the organelle marker proteins (filled shapes). Inverted triangles, vacuolar membrane and vacuolar ABC transporters; diamonds, PM and PM ABC transporters; circles, mitochondria/plastids; star, mitochondrial ABC transporter; diagonal crosses, vacuolar sorting receptors; horizontal cross, V-ATPase subunit V0a; triangles, Golgi apparatus, GMT1, and GMT2; squares, ER.

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