In vivo expression pattern of a plant K + channel β subunit protein

Abstract

Studies described in this report employed an antibody generated against a plant ( Arabidopsis thaliana) K + channel β subunit polypeptide (`KAB1'). Immunoblot detection with anti-KAB1 as a probe of native protein isolated from a broad range of plant species and size-fractionated on SDS-PAGE indicated the presence of KAB1 homologs in some, but not all species tested. Immunocytochemical studies (at the subcellular level) were also undertaken on A. thaliana leaf and root tissue preparations. Hydropathy analysis of the KAB1 sequence suggests that it is a hydrophilic polypeptide, and immunoblot analysis of SDS-PAGE size-fractionated native protein isolated from A. thaliana plants identified KAB1 in a soluble protein fraction. However, electron micrograph analysis of immunogold staining with anti-KAB1 revealed native KAB1 as present in membrane systems. KAB1 protein was present in the plasmalemma and tonoplast of both root and leaf cells, and the mitochondrial and chloroplast inner envelope of leaf cells. These results, along with covalent cross-linking studies, suggested that the soluble KAB1 polypeptide associates with integral membrane proteins such as the pore-forming ( α) subunit of K + channels in situ. Localization of KAB1 protein in root and leaf membranes suggests that this K + channel β subunit may associate with more than one type of α subunit in vivo. In vitro protein:protein interaction studies confirm the ability of KAB1 to form protein complexes with multiple plant K + channel α subunits. Immunogold staining patterns of membrane-associated KAB1 indicated that the native polypeptide is present in regularly-spaced individual protein complexes. This regular staining pattern allowed for theoretical estimations of K + channel density in plant cell membranes. It was concluded from such structural analyses that these ion channels are present in extremely low copy number in plant membranes; a finding consistent with prior estimations from ion conductance measurements. The structural characterization of K + channels afforded by these electron micrograph studies is significant in that it represents the first subcellular immunocytochemical localization of K + channel proteins native to either plant or animal membranes.