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Cellulose Synthesis on Isolated Tubulin-Containing Granules from Plasma Membranes of Azuki Bean Epicotyls

小豆上胚轴质膜分离含微管蛋白颗粒的纤维素合成

基本信息

批准号：
08454257
负责人：
MIZUNO Koichi
金额：
$ 5.06万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Until recently, tubulin was always considered exclusively to be a major constituent of cytoplasmic microtubules, which participatein a wide variety of processes in plant development. However, the presence has been confirmed of a unique tubulin in the plasma membranes of higher plants and was assumed to have an important role for cell elongation. Cortical Mts are considered to control the orientation of cellulose microfibrils in higher plant cells since MTs are often aligned parallel to newly desposited microfibrils. However, the mechanism whereby cortical MTs control the orientation of cellulose microfibrils has not been clarified. It has been postulated that cellulose microfibrils are assembled by terminal complexes in association with the plasma membrane. Terminal complexes have been observed in freeze-fracture replicas of the plasma membranes of various cells, forming rosettes of about 25 nm in diameter. The rosette structure consists of six granules of 8-9 nm in diameter. In higher … More plant cells, the isolation and identification of terminal complexes has not yet been successful, although some possible models for the relation of MTs and cellulose synthase have been presented.In a detergent-solubilized preparation of plasma membranes from epicotyls of azuki bean, tubulin was shown to be integrated in granules of about 10 nm in diameter. The size of these granules was similar to that of the granules that form the rossette structures. The method was developed to isolate these granules using antibodies against tubulin successfully. Asnalysis on SDS-PAGE indicated that the granules consisted of several proteins, 33,42 and 120 kD,respectively, including tubulin as the most abundant constituent. Using specific antibodies against 33 kD protein, in vitro synthesis of cellulose was inhibited entirely and the immunoelectron microscopy indicated that 33 kD protein is integrated in these granules. Since the 33 kD protein binds ^<32>P-labeled UDP-glucose, this protein was estimated to be important constituent of complexes. The cloning of gene coding 33kD protein was performed and the identification of amino acid sequence was also accomplished. Less

直到最近，微管蛋白一直被认为是细胞质微管的主要成分，参与植物发育的各种过程。然而，高等植物的质膜中存在一种独特的微管蛋白，并被认为对细胞伸长起重要作用。皮层Mts被认为控制着高等植物细胞中纤维素微原纤维的方向，因为Mts通常与新沉积的微原纤维平行排列。然而，皮层MTs控制纤维素微原纤维取向的机制尚不清楚。据推测，纤维素微原纤维是由与质膜结合的末端复合物组装而成的。在各种细胞的质膜冷冻断裂复制品中观察到末端络合物，形成直径约25 nm的莲座。莲座结构由6个直径为8-9纳米的颗粒组成。在高等植物细胞中，虽然已经提出了一些可能的MTs与纤维素合酶关系的模型，但末端复合物的分离和鉴定尚未成功。在一种用洗涤剂溶解的方法制备的红豆上胚轴质膜中，微管蛋白被整合在直径约10纳米的颗粒中。这些颗粒的大小与形成玫瑰花结构的颗粒相似。该方法利用抗微管蛋白抗体成功分离了这些颗粒。SDS-PAGE分析表明，这些颗粒分别由33、42和120 kD的蛋白质组成，其中微管蛋白含量最高。使用针对33 kD蛋白的特异性抗体，纤维素的体外合成完全被抑制，免疫电镜显示33 kD蛋白整合在这些颗粒中。由于33 kD蛋白结合^<32> p标记的udp -葡萄糖，估计该蛋白是复合物的重要组成部分。克隆了编码33kD蛋白的基因，并鉴定了其氨基酸序列。少