Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells.
KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels. They were biochemically characterized after expression in insect cells using recombinant baculoviruses. The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques. The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells. The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides. Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches. Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.
Urbach
S
S
Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, UMR 5004, Agro-M/CNRS/INRA/UMII, 34060 Montpellier Cedex 1, France.
Chérel
I
I
Sentenac
H
H
Gaymard
F
F
D000818:Animals; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007313:Insecta / Q000166:cytology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D020798:Two-Hybrid System Techniques
D029681:Arabidopsis Proteins; D002457:Cell Extracts; C501210:KAT1 protein, Arabidopsis; D010940:Plant Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C074500:AKT1 protein, Arabidopsis