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

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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