Alternative Splicing-Mediated Targeting of the Arabidopsis GLUTAMATE RECEPTOR3.5 to Mitochondria Affects Organelle Morphology

Abstract

Since the discovery of 20 genes encoding for putative ionotropic glutamate receptors in the Arabidopsis (Arabidopsis thaliana) genome, there has been considerable interest in uncovering their physiological functions. For many of these receptors, neither their channel formation and/or physiological roles nor their localization within the plant cells is known. Here, we provide, to our knowledge, new information about in vivo protein localization and give insight into the biological roles of the so-far uncharacterized Arabidopsis GLUTAMATE RECEPTOR3.5 (AtGLR3.5), a member of subfamily 3 of plant glutamate receptors. Using the pGREAT vector designed for the expression of fusion proteins in plants, we show that a splicing variant of AtGLR3.5 targets the inner mitochondrial membrane, while the other variant localizes to chloroplasts. Mitochondria of knockout or silenced plants showed a strikingly altered ultrastructure, lack of cristae, and swelling. Furthermore, using a genetically encoded mitochondria-targeted calcium probe, we measured a slightly reduced mitochondrial calcium uptake capacity in the knockout mutant. These observations indicate a functional expression of AtGLR3.5 in this organelle. Furthermore, AtGLR3.5-less mutant plants undergo anticipated senescence. Our data thus represent, to our knowledge, the first evidence of splicing-regulated organellar targeting of a plant ion channel and identify the first cation channel in plant mitochondria from a molecular point of view.

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

“…Indeed, different systems may contribute to mitochondrial Ca 2+ dynamics, but the physical players involved are still largely unknown. Recently, the glutamate receptor 3.5 (GLR3.5) was identified as a putative plant mitochondrial cation transporter (Teardo et al, 2015). However, despite Ca 2+ being a potential substrate, no pronounced changes in mitochondrial Ca 2+ dynamics could be detected.…”

Signalling via glutamate and GLRs in Arabidopsis thaliana

“…No inducible Ca 2+ fluxes were detected until the addition of the AMPA receptor regulatory protein 'TARP g-2' (Tapken and Hollmann 2008). This observation could help to explain why electrophysiological studies using heterologous expressions are still hard to conduct and investigations often fail (Vincill et al 2013;Teardo et al 2015). It appears that the GLR complex, which contains several different AtGLR subunits, is highly sophisticated and needs auxiliary proteins and an accurate composition to work correctly.…”

“…Soon after their discovery, it became clear that all GLRs are expressed in Arabidopsis, with at least three of them (AtGLR2.5, AtGLR3.4 and AtGLR3.5) occurring as mRNA splice variants (Chiu et al 2002;Meyerhoff et al 2005;Teardo et al 2015). The existence of different spliced forms of mRNAs allows an even broader spectrum of involvements and functions for these receptors by multiplying significantly possible receptor complex combinations (see 'Molecular structure').…”

“…Noteworthy, a recent study by Teardo et al (2015) demonstrates the existence of alternative mRNA splice variants of AtGLRs and their impact on the integration of the same receptor in different membranes. It could be shown that AtGLR3.5 contains an N-terminal signal sequence for mitochondria that is removed in another isoform.…”

“…Actually, this leads to a localisation of one isoform at the inner mitochondrial membrane where its C-terminus faces the matrix and its ligandbinding sites the cytosol. The other isoform is situated in chloroplasts (Teardo et al 2015). The phenomenon of alternative splicing in GLRs could amplify the flexibility of some glutamate receptors in respect to potential sites of action and signal modulations.…”