Physical Association of KAB1 with Plant K + Channel a Subunits

Abstract

K+ channel proteins contain four a subunits that align along a central axis perpendicular to membranes and form an ion-conducting pore. Recent work with K+ channels native to animal membranes has shown that at least some members of this protein family also have four p subunits. These structural components of the holoenzyme each form tight associations with the cytoplasmic portion of an a subunit. We have cloned an Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid identity with animal K+ channel p subunits. In this study, we provide experimental evidence that the KABl polypeptide forms a tight physical association with the Arabidopsis K+ channel a subunit, KAT1. An affinity-purified KABl fusion protein was immobilized to a support resin and shown to sequester selectively the KATl polypeptide. In addition, polyclonal antibodies raised against KABl were shown to immunoprecipitate the KATl polypeptide as a KAT1-KAB1 protein complex. lmmunoblot analysis demonstrated that KABl is expressed in Arabidopsis seedlings and is present in both membrane and soluble protein fractions. The presence of KABl (a soluble polypeptide) in both soluble and membrane protein fractions suggests that a portion of the total amount of native KABl is associated with an integral membrane protein, such as KAT1. The presence of KABl in crude protein fractions prepared from different Arabidopsis plant organs was evaluated. High levels of KABl protein were present in flowers, roots, and leaves. lmmunoblot analysis of protein extracts prepared from broad bean leaves indicated that the KABl expression leve1 was 80-fold greater in guard cells than in mesophyll cells. Previous studies of the in situ transcription pattern of KATl in Arabidopsis indicated that this a subunit is abundantly present in leaves and, within the leaf, exclusively present in guard cells. Thus, KABl was determined to be expressed in plant organs (leaves) and cell types (guard cells) that are sites of KATl expression in the plant. The in situ expression pattern of KABl suggests that it may associate with more than one type of K+ channel a subunit. Sequence analysis indicates that KABl may function in plant K+ channels as an oxidoreductase. It is postulated that p subunits native to animal K+ channels act as regulatory subunits through pyridine nucleotide-linked reduction of a polypeptides. Although the KABl primary structure is substantially different from that of animal p subunits, amino acid motifs critical for this catalytic activity are retained in the plant p subunit.