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

Abstract

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 In order to locate or identify proteins within subcellular compartments, organelles need to be isolated in high purity. This can be achieved through density gradient fractionation in aqueous media using sucrose or Percoll as a matrix. Homogenized material or crude organelle preparations are applied to density gradients, and fractions are collected after centrifugation. The distribution profiles of organelles in these partially enriched fractions are characterized, for example, by assays of organelle-specific enzymes (6, 7). Proteins displaying a distribution profile similar to those of organelle marker proteins can then be assigned to this subcellular compartment. This technique has for a long time been used in plant biology to study enzyme activities associated with organelle-enriched fractions (8). In recent decades, the number of proteins identified within cellular compartments has significantly increased, in particular because of improvements in mass-spectrometrybased proteomic methods. Among others, the human centrosome proteome (9), the mouse organelle proteome (10), and membrane proteins from Arabidopsis (11-13) have been characterized.

Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein

“…Many amino acids are located in both the cytoplasm and the vacuole (Riens et al, 1991;Weiner and Heldt, 1992;Winter et al, 1992;Krueger et al, 2011;Arrivault et al, 2014), and it is likely that the cytoplasmic pool is more rapidly labeled than the vacuolar pool (Szecowka et al, 2013). There may also be pools in nonphotosynthetic cells that are not rapidly labeled with 13 CO 2 .…”

Genome-Wide Association Mapping Reveals That Specific and Pleiotropic Regulatory Mechanisms Fine-Tune Central Metabolism and Growth in Arabidopsis

“…Alternatively, fumarase activity might be modified in a manner that our standardized assays do not detect (e.g., via a change in substrate affinity). The vast majority of the Mal and Fum in plant cells is located in the vacuole (Krueger et al, 2011;Arrivault et al, 2014). The lack of effect of the FUM1 enzyme activity QTL on the levels of these metabolites, and the association of Mal and Fum metabolite QTL with FUM2, indicate that cytosolic fumarase determines the vacuolar levels of Mal and Fum.…”

Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling

“…Therefore, the aim of this work was to use density gradient centrifugation to separate organellar membranes and their associated proteins and to systematically search for altered protein distributions in plants with mutations in ␤-subunits of the adaptor complexes AP-3 and AP-4 (12,14). Density-based separation of organelle membranes is a well-established technique to identify protein complexes and protein distribution in cells (15)(16)(17)(18)(19) and has recently been extended to study the subcellular distribution of metabolite-protein complexes to organelles (20). This simultaneous separation and enrichment of endomembranes followed by the identification of their protein contents gives a comprehensive distribution profile of proteins and in addition, is sensitive to detect differences in protein distributions.…”