Dissecting the Subcellular Compartmentation of Proteins and Metabolites in Arabidopsis Leaves Using Non-aqueous Fractionation *

Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin–Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite–protein analysis on a single tissue sample.

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