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Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins

Plant cells are unique as they carry two organelles of endosymbiotic origin, namely mitochondria and chloroplasts (plastids) which have specific but partially overlapping functions, e. g., in energy and redox metabolism. Despite housing residual genomes of limited coding capacity, most of their prot...

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Autores principales: Sharma, Mayank, Bennewitz, Bationa, Klösgen, Ralf Bernd
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160753/
https://www.ncbi.nlm.nih.gov/pubmed/30298079
http://dx.doi.org/10.3389/fpls.2018.01350
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author Sharma, Mayank
Bennewitz, Bationa
Klösgen, Ralf Bernd
author_facet Sharma, Mayank
Bennewitz, Bationa
Klösgen, Ralf Bernd
author_sort Sharma, Mayank
collection PubMed
description Plant cells are unique as they carry two organelles of endosymbiotic origin, namely mitochondria and chloroplasts (plastids) which have specific but partially overlapping functions, e. g., in energy and redox metabolism. Despite housing residual genomes of limited coding capacity, most of their proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and need to be transported “back” into the respective target organelle. While transport is in most instances strictly monospecific, a group of proteins carries “ambiguous” transit peptides mediating transport into both, mitochondria and plastids. However, such dual targeting is often disputed due to variability in the results obtained from different experimental approaches. We have therefore compared and evaluated the most common methods established to study protein targeting into organelles within intact plant cells. All methods are based on fluorescent protein technology and live cell imaging. For our studies, we have selected four candidate proteins with proven dual targeting properties and analyzed their subcellular localization in vivo utilizing four different methods (particle bombardment, protoplast transformation, Agrobacterium infiltration, and transgenic plants). Though using identical expression constructs in all instances, a given candidate protein does not always show the same targeting specificity in all approaches, demonstrating that the choice of method is important, and depends very much on the question to be addressed.
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spelling pubmed-61607532018-10-08 Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins Sharma, Mayank Bennewitz, Bationa Klösgen, Ralf Bernd Front Plant Sci Plant Science Plant cells are unique as they carry two organelles of endosymbiotic origin, namely mitochondria and chloroplasts (plastids) which have specific but partially overlapping functions, e. g., in energy and redox metabolism. Despite housing residual genomes of limited coding capacity, most of their proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and need to be transported “back” into the respective target organelle. While transport is in most instances strictly monospecific, a group of proteins carries “ambiguous” transit peptides mediating transport into both, mitochondria and plastids. However, such dual targeting is often disputed due to variability in the results obtained from different experimental approaches. We have therefore compared and evaluated the most common methods established to study protein targeting into organelles within intact plant cells. All methods are based on fluorescent protein technology and live cell imaging. For our studies, we have selected four candidate proteins with proven dual targeting properties and analyzed their subcellular localization in vivo utilizing four different methods (particle bombardment, protoplast transformation, Agrobacterium infiltration, and transgenic plants). Though using identical expression constructs in all instances, a given candidate protein does not always show the same targeting specificity in all approaches, demonstrating that the choice of method is important, and depends very much on the question to be addressed. Frontiers Media S.A. 2018-09-19 /pmc/articles/PMC6160753/ /pubmed/30298079 http://dx.doi.org/10.3389/fpls.2018.01350 Text en Copyright © 2018 Sharma, Bennewitz and Klösgen. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Sharma, Mayank
Bennewitz, Bationa
Klösgen, Ralf Bernd
Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title_full Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title_fullStr Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title_full_unstemmed Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title_short Dual or Not Dual?—Comparative Analysis of Fluorescence Microscopy-Based Approaches to Study Organelle Targeting Specificity of Nuclear-Encoded Plant Proteins
title_sort dual or not dual?—comparative analysis of fluorescence microscopy-based approaches to study organelle targeting specificity of nuclear-encoded plant proteins
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160753/
https://www.ncbi.nlm.nih.gov/pubmed/30298079
http://dx.doi.org/10.3389/fpls.2018.01350
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