Abstract
Calmodulins (CaMs) are important mediators of Ca(2+) signals that are found ubiquitously in all eukaryotic organisms. Plants contain a unique family of calmodulin-like proteins (CMLs) that exhibit greater sequence variance compared to canonical CaMs. The Arabidopsis thaliana proteins AtCML4 and AtCML5 are members of CML subfamily VII and possess a CaM domain comprising the characteristic double pair of EF-hands, but they are distinguished from other members of this subfamily and from canonical CaMs by an N-terminal extension of their amino acid sequence. Transient expression of yellow fluorescent protein-tagged AtCML4 and AtCML5 under a 35S-promoter in Nicotiana benthamiana leaf cells revealed a spherical fluorescence pattern. This pattern was confirmed by transient expression in Arabidopsis protoplasts under the native promoter. Co-localization analyses with various endomembrane marker proteins suggest that AtCML4 and AtCML5 are localized to vesicular structures in the interphase between Golgi and the endosomal system. Further studies revealed AtCML5 to be a single-pass membrane protein that is targeted into the endomembrane system by an N-terminal signal anchor sequence. Self-assembly green fluorescent protein and protease protection assays support a topology with the CaM domain exposed to the cytosolic surface and not the lumen of the vesicles, indicating that AtCML5 could sense Ca(2+) signals in the cytosol. Phylogenetic analysis suggests that AtCML4 and AtCML5 are closely related paralogues originating from a duplication event within the Brassicaceae family. CML4/5-like proteins seem to be universally present in eudicots but are absent in some monocots. Together these results show that CML4/5-like proteins represent a flowering plant-specific subfamily of CMLs with a potential function in vesicle transport within the plant endomembrane system.
Highlights
Plants have evolved to adapt to their specific habitats, result- exposed to rapid and often unpredictable abiotic and biotic ing in optimized growth and development
AtCML4 and AtCML5 are endomembrane system localized calmodulin-like protein (CML) | Page 5 of 12 and AtCML5 in Arabidopsis protoplasts controlled by their respective endogenous promoters results in a similar fluorescence signal pattern (Supplementary Fig. S1B), the observed signal in tobacco protoplasts is not an artefact caused by 35S-promoter driven overexpression
CMLs have been identified in compartments other than the cytoplasm, including two CMLs targeted into mitochondria and peroxisomes by means of N- or C-terminal sequence extensions (Chigri et al, 2012; Bender and Snedden, 2013)
Summary
Plants have evolved to adapt to their specific habitats, result- exposed to rapid and often unpredictable abiotic and biotic ing in optimized growth and development. Proteins that allows them to react to certain stimuli on a cellular level (reviewed in Kudla et al, 2010). Within this context, Ca2+ is a secondary messenger that plays an important role in mediating environmental cues into an appropriate cellular response. Many different biotic and abiotic stimuli are known to cause temporal and spatial changes in intracellular Ca2+ concentration that are recognized by specific Ca2+ sensors (DeFalco et al, 2010; Dodd et al, 2010; Kudla et al, 2010). CaM has been shown to affect many different cellular processes, including changes in gene expression, activation of ion channels, initiation of phosphorylation cascades, and the direct alteration of metabolic enzymes (reviewed in White and Broadley, 2003; Yang and Poovaiah, 2003; Bouche et al, 2005)
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