Tang H, Vasconcelos A C, Berkowitz G A
Plant Science Department, Cook College, Rutgers, State University of New Jersey, New Brunswick 08903, USA.
Plant Cell. 1996 Sep;8(9):1545-53. doi: 10.1105/tpc.8.9.1545.
K+ channel proteins contain four alpha 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 beta subunits. These structural components of the holoenzyme each form tight associations with the cytoplasmic portion of an alpha subunit. We have cloned an Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid identity with animal K+ channel beta subunits. In this study, we provide experimental evidence that the KAB1 polypeptide forms a tight physical association with the Arabidopsis K+ channel alpha subunit, KAT1. An affinity-purified KAB1 fusion protein was immobilized to a support resin and shown to sequester selectively the KAT1 polypeptide. In addition, polyclonal antibodies raised against KAB1 were shown to immunoprecipitate the KAT1 polypeptide as a KAT1-KAB1 protein complex. Immunoblot analysis demonstrated that KAB1 is expressed in Arabidopsis seedings and is present in both membrane and soluble protein fractions. The presence of KAB1 (a soluble polypeptide) in both soluble and membrane protein fractions suggests that a portion of the total amount of native KAB1 is associated with an integral membrane protein, such as KAT1. The presence of KAB1 in crude protein fractions prepared from different Arabidopsis plant organs was evaluated. High levels of KAB1 protein were present in flowers, roots, and leaves. Immunoblot analysis of protein extracts prepared from broad bean leaves indicated that the KAB1 expression level was 80-fold greater in guard cells than in mesophyll cells. Previous studies of the in situ transcription pattern of KAT1 in Arabidopsis indicated that this alpha subunit is abundantly present in leaves and, within the leaf, exclusively present in guard cells. Thus, KAB1 was determined to be expressed in plant organs (leaves) and cell types (guard cells) that are sites of KAT1 expression in the plant. The in situ expression pattern of KAB1 suggests that it may associate with more than one type of K+ channel alpha subunit. Sequence analysis indicates that KAB1 may function in plant K+ channels as an oxidoreductase. It is postulated that beta subunits native to animal K+ channels act as regulatory subunits through pyridine nucleotide-linked reduction of alpha polypeptides. Although the KAB1 primary structure is substantially different from that of animal beta subunits, amino acid motifs critical for this catalytic activity are retained in the plant beta subunit.
钾离子通道蛋白包含四个α亚基，这些亚基沿着垂直于细胞膜的中心轴排列并形成一个离子传导孔。最近对动物细胞膜原生钾离子通道的研究表明，这个蛋白质家族的至少一些成员还具有四个β亚基。全酶的这些结构成分各自与一个α亚基的胞质部分紧密结合。我们克隆了拟南芥的一个cDNA（KAB1），它编码的一种多肽与动物钾离子通道β亚基有49%的氨基酸同源性。在本研究中，我们提供了实验证据，证明KAB1多肽与拟南芥钾离子通道α亚基KAT1形成紧密的物理结合。一种亲和纯化的KAB1融合蛋白被固定在支持树脂上，并显示能选择性地螯合KAT1多肽。此外，针对KAB1产生的多克隆抗体显示能将KAT1多肽作为KAT1 - KAB1蛋白复合物进行免疫沉淀。免疫印迹分析表明，KAB1在拟南芥幼苗中表达，并且存在于膜蛋白和可溶性蛋白组分中。KAB1（一种可溶性多肽）同时存在于可溶性和膜蛋白组分中，这表明天然KAB1总量的一部分与一种整合膜蛋白（如KAT1）相关联。评估了从拟南芥不同植物器官制备的粗蛋白组分中KAB1的存在情况。花、根和叶中存在高水平的KAB1蛋白。对蚕豆叶制备的蛋白提取物进行免疫印迹分析表明，保卫细胞中KAB1的表达水平比叶肉细胞高80倍。先前对拟南芥中KAT1原位转录模式的研究表明，这个α亚基大量存在于叶片中，并且在叶片内仅存在于保卫细胞中。因此，确定KAB1在植物中KAT1表达的植物器官（叶片）和细胞类型（保卫细胞）中表达。KAB1的原位表达模式表明它可能与不止一种类型的钾离子通道α亚基相关联。序列分析表明，KAB1在植物钾离子通道中可能作为一种氧化还原酶发挥作用。据推测，动物钾离子通道的天然β亚基通过吡啶核苷酸连接的α多肽还原作用作为调节亚基发挥作用。虽然KAB1的一级结构与动物β亚基有很大不同，但对于这种催化活性至关重要的氨基酸基序在植物β亚基中得以保留。
