IN-VIVO EXPRESSION PATTERN OF A PLANT K-SUBUNIT PROTEIN( CHANNEL BETA)

Studies described in this report employed an antibody generated agains t a plant (Arabidopsis thaliana) K+ channel beta subunit polypeptide ( 'KAB1'). Immunoblot detection with anti-KAB1 as a probe of native prot ein 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 lev el) were also undertaken on A. thaliana leaf and root tissue preparati ons. Hydropathy analysis of the KAB1 sequence suggests that it is a hy drophilic polypeptide, and immunoblot analysis of SDS-PAGE size-fracti onated 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 t onoplast of both root and leaf cells, and the mitochondrial and chloro plast inner envelope of leaf cells. These results, along with covalent cross-linking studies, suggested that the soluble KAB1 polypeptide as sociates with integral membrane proteins such as the pore-forming (alp ha) subunit of K+ channels in situ. Localization of KAB1 protein in ro ot and leaf membranes suggests that this K+ channel beta subunit may a ssociate with more than one type of alpha subunit in vivo. In vitro pr otein:protein interaction studies confirm the ability of KAB1 to form protein complexes with multiple plant K+ channel alpha subunits. Immun ogold staining patterns of membrane-associated KABI indicated that the native polypeptide is present in regularly-spaced individual protein complexes. This regular staining pattern allowed for theoretical estim ations 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 wit h 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 imm unocytochemical localization of K+ channel proteins native to either p lant or animal membranes. (C) 1998 Elsevier Science Ireland Ltd. All r ights reserved.