title: The calmodulin-like proteins AtCML4 and AtCML5 are single-pass membrane proteins targeted to the endomembrane system by an N-terminal signal anchor sequence.
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.
authors.affiliation: Department of Biology I, Faculty of Biology, LMU Munich, Großhaderner Straße 2-4, D-82152 Planegg, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Ruge
authors.affiliation: Department of Biology I, Faculty of Biology, LMU Munich, Großhaderner Straße 2-4, D-82152 Planegg, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Flosdorff
authors.affiliation: Department for Applied Bioinformatics, Institute for Cell Biology and Neuroscience, Goethe-University Frankfurt, Max-von-Laue-Straße 13, 60438 Frankfurt am Main, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Ebersberger
authors.affiliation: Department of Biology I, Faculty of Biology, LMU Munich, Großhaderner Straße 2-4, D-82152 Planegg, Germany Center for Integrated Protein Science (Munich) at the Department of Biology I, Faculty of Biology, LMU Munich, D-81377 Munich, Germany.
authors.forename: Fatima
authors.initials: F
authors.lastname: Chigri
authors.affiliation: Department of Biology I, Faculty of Biology, LMU Munich, Großhaderner Straße 2-4, D-82152 Planegg, Germany Center for Integrated Protein Science (Munich) at the Department of Biology I, Faculty of Biology, LMU Munich, D-81377 Munich, Germany vothknecht@bio.lmu.de.
authors.forename: Ute C
authors.initials: UC
authors.lastname: Vothknecht
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D030821:Plants, Genetically Modified / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D016415:Sequence Alignment; D014026:Nicotiana / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; C000625168:CML4 protein, Arabidopsis; C000625169:CML5 protein, Arabidopsis; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals
keywords: Arabidopsis; Golgi; calcium signalling; calmodulin; endomembrane system; endosomes; vesicle transport.