Vacuolar membrane localization of the Arabidopsis 'two-pore' K+ channel KCO1.
Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.
Czempinski
Katrin
K
Universität Potsdam, Institut für Biochemie und Biologie, Golm, Germany. czempins@rz.uni-potsdam.de
Frachisse
Jean-Marie
JM
Maurel
Christophe
C
Barbier-Brygoo
Helene
H
Mueller-Roeber
Bernd
B
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000502:physiology*; D029681:Arabidopsis Proteins; D002460:Cell Line; D017124:Conserved Sequence / Q000235:genetics; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024683:Potassium Channels, Tandem Pore Domain; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000235:genetics; D014617:Vacuoles / Q000378:metabolism*
D029681:Arabidopsis Proteins; C503455:KCO1 protein, Arabidopsis; D008164:Luminescent Proteins; D010940:Plant Proteins; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005966:Glucuronidase