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

Mayank Sharma,Bationa Bennewitz,Ralf Bernd Klösgen

doi:10.3389/fpls.2018.01350

Mayank Sharma, Bationa Bennewitz + Show 1 more

Open Access

https://doi.org/10.3389/fpls.2018.01350

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Abstract

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.

Highlights

As a consequence of the endosymbiotic gene transfer, the vast majority of proteins from mitochondria and chloroplasts are encoded in the nuclear genome, and synthesized in the cytosol of the eukaryotic plant cell (Martin and Herrmann, 1998; Bock and Timmis, 2008)
For the comparative analysis of the most common in vivo approaches used to study intracellular protein targeting in plants, we have focused on four candidate proteins from A. thaliana, namely GCS (Glycine cleavage system subunit-H 1, At2g35370), GrpE, EF-Tu, and PDF
A good example for such variability is GrpE/eYFP which shows dual targeting in all transient assays described here but accumulates in transgenic plants solely in mitochondria

Summary

Introduction

As a consequence of the endosymbiotic gene transfer, the vast majority of proteins from mitochondria and chloroplasts are encoded in the nuclear genome, and synthesized in the cytosol of the eukaryotic plant cell (Martin and Herrmann, 1998; Bock and Timmis, 2008). Subsequent transport into the respective target organelle is mediated by N-terminal extensions, called presequences or transit peptides, which comprise all of the information for organelle targeting and transport. In most instances, this process is monospecific, i.e., a given protein is transported solely into a single type of organelle. Approaches to Study Protein Targeting Specificity dual targeting properties and can be imported into both, mitochondria and plastids (Peeters and Small, 2001). In many cases, such dual targeting is mediated by “ambiguous” transit peptides, which are capable of interacting with the protein import machineries of both endosymbiotic organelles (summarized in Carrie and Small, 2013). Current estimates assume that ∼5% of the proteins from mitochondria and plastids possess dual targeting properties (Mitschke et al, 2009; Baudisch et al, 2014)

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