Subcellular proteomics—where cell biology meets protein chemistry

Millar, A. Harvey; Taylor, Nicolas L.

doi:10.3389/fpls.2014.00055

Cited by 20 publications

(13 citation statements)

References 27 publications

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“…While protein data can often be interpreted in context of subcellular compartments due to characteristic amino acid sequences and enrichment techniques (Millar and Taylor, 2014), accompanying dynamics of the metabolome cannot be resolved equivalently. Here, we present a method which is capable of resolving the subcellular metabolome in a high-throughput benchtop manner by correlating metabolite abundances with compartment-specific marker enzyme activities.…”

Section: Discussionmentioning

confidence: 99%

A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome

2016

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Although compartmentation is a key feature of eukaryotic cells, biological research is frequently limited by methods allowing for the comprehensive subcellular resolution of the metabolome. It has been widely accepted that such a resolution would be necessary in order to approximate cellular biochemistry and metabolic regulation, yet technical challenges still limit both the reproducible subcellular fractionation and the sample throughput being necessary for a statistically robust analysis. Here, we present a method and a detailed protocol which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular localization. The presented benchtop method aims at unraveling subcellular metabolome dynamics in a precise and statistically robust manner using a relatively small amount of tissue material. The method is based on the separation of cellular fractions via density gradients consisting of organic, non-aqueous solvents. By determining the relative distribution of compartment-specific marker enzymes together with metabolite profiles over the density gradient it is possible to estimate compartment-specific metabolite concentrations by correlation. To support this correlation analysis, a spreadsheet is provided executing a calculation algorithm to determine the distribution of metabolites over subcellular compartments. The calculation algorithm performs correlation of marker enzyme activity and metabolite abundance accounting for technical errors, reproducibility and the resulting error propagation. The method was developed, tested and validated in three natural accessions of Arabidopsis thaliana showing different ability to acclimate to low temperature. Particularly, amino acids were strongly shuffled between subcellular compartments in a cold-sensitive accession while a cold-tolerant accession was characterized by a stable subcellular metabolic homeostasis. Finally, we conclude that subcellular metabolome analysis is essential to unambiguously unravel regulatory strategies being involved in plant-environment interactions.

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Section: Discussionmentioning

confidence: 99%

A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome

2016

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“…Our results indicate that different subcellular organelles potentially contribute differentially to the reprogramming process, yielding a stable metabolic homeostasis only under certain preconditions. While it might not be surprising that different cellular organelles contribute differentially to metabolic regulation due to their different pH, volumes or proteomes (Ito et al 2014 ; Joyard et al 2010 ; Lunn 2007 ; Millar and Taylor 2014 ), the quantification of these contributions remains challenging. One example indicating the regulatory complexity on a subcellular and molecular level is the finding that vacuolar sugar carriers contribute differentially to metabolic reprogramming in different environmental conditions (Klemens et al 2013 ).…”

Section: Discussionmentioning

confidence: 99%

Approximating the stabilization of cellular metabolism by compartmentalization

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2016

Theory Biosci.

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Biochemical regulation in compartmentalized metabolic networks is highly complex and non-intuitive. This is particularly true for cells of higher plants showing one of the most compartmentalized cellular structures across all kingdoms of life. The interpretation and testable hypothesis generation from experimental data on such complex systems is a challenging step in biological research and biotechnological applications. While it is known that subcellular compartments provide defined reaction spaces within a cell allowing for the tight coordination of complex biochemical reaction sequences, its role in the coordination of metabolic signals during metabolic reprogramming due to environmental fluctuations is less clear. In the present study, we numerically analysed the effects of environmental fluctuations in a subcellular metabolic network with regard to the stability of an experimentally observed steady state in the genetic model plant Arabidopsis thaliana. Applying a method for kinetic parameter normalization, several millions of probable enzyme kinetic parameter constellations were simulated and evaluated with regard to the stability information of the metabolic homeostasis. Information about the stability of the metabolic steady state was derived from real parts of eigenvalues of Jacobian matrices. Our results provide evidence for a differential stabilizing contribution of different subcellular compartments. We could identify stabilizing and destabilizing network components which we could classify according to their subcellular localization. The findings prove that a highly dynamic interplay between intracellular compartments is preliminary for an efficient stabilization of a metabolic homeostasis after environmental perturbation. Further, our results provide evidence that feedback-inhibition originating from the cytosol and plastid seem to stabilize the sucrose homeostasis more efficiently than vacuolar control. In summary, our results indicate stabilizing and destabilizing network components in context of their subcellular organization.Electronic supplementary materialThe online version of this article (doi:10.1007/s12064-016-0225-y) contains supplementary material, which is available to authorized users.

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“…Therefore, the crop stress response should be analyzed at a cellular or subcellular level, integrated with studies on whole plants, organs or tissues, to discriminate the specific responses of different cell types to abiotic stress. At present, cell or subcellular proteomic studies focus on relatively abundant, or easily isolated homogenous compartments (e.g., plastids, mitochondria, peroxisomes, and nuclei) mainly in Arabidopsis (Tanz et al, 2013 ), but also in rice, wheat, barley, maize (Huang et al, 2013 ; Millar and Taylor, 2014 ; Hu et al, 2015 ).…”

Section: Dissecting Cell or Tissue Specific Stress Responsementioning

confidence: 99%