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Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants.

ABSTRACT: Fluorescent protein (FP) tagging approaches are widely used to determine the subcellular location of plant proteins. Here we give a brief overview of FP approaches, highlight potential technical problems, and discuss what to consider when designing FP/protein fusion constructs and performing transformation assays. We analyze published FP tagging data sets along with data from proteomics studies collated in SUBA3, a subcellular location database for Arabidopsis proteins, and assess the reliability of these data sets by comparing them. We also outline the limitations of the FP tagging approach for defining protein location and investigate multiple localization claims by FP tagging. We conclude that the collation of localization datasets in databases like SUBA3 is helpful for revealing discrepancies in location attributions by different techniques and/or by different research groups.

SUBMITTER: Tanz SK

PROVIDER: S-EPMC3690342 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants.

Tanz Sandra K SK Castleden Ian I Small Ian D ID Millar A Harvey AH

Frontiers in plant science 20130624

Fluorescent protein (FP) tagging approaches are widely used to determine the subcellular location of plant proteins. Here we give a brief overview of FP approaches, highlight potential technical problems, and discuss what to consider when designing FP/protein fusion constructs and performing transformation assays. We analyze published FP tagging data sets along with data from proteomics studies collated in SUBA3, a subcellular location database for Arabidopsis proteins, and assess the reliabilit ...[more]

PMID: 23805149

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Project description:BackgroundIntrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.ResultsWhen expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.ConclusionIn plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

Dataset Information

Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants.

Publications

Fluorescent protein tagging as a tool to define the subcellular distribution of proteins in plants.

Similar Datasets

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