Lynne M. Roberts

Protein biosynthetic capacity in the endosperm tissue of ripening castor bean seeds

Endosperm tissue was excised from Ricinus communis plants at different stages during seed maturation. The various stages were characterized on the basis of total RNA, protein and lipid content. Polyadenylated RNA was recovered from the total RNA by affinity chromatography on oligo(dT)cellulose. With the exception of that isolated from dry seeds, this poly(A+) RNA actively programmed protein synthesis in cell-free systems containing either wheat germ S30 extracts or nuclease treated rabbit reticulocyte lysates at each developmental stage examined. Translational products were separated electrophoretically and were visualized by fluorography. The capacity to synthesize protein was also estimated during ‘in vivo’ labelling studies. Developmental changes in the capacity of maturing endosperm tissue to synthesize a characteristic protein, R. communis agglutinin, were followed by immunoprecipitating this protein from the total ‘in vitro’ products synthesized at various stages. Endoplasmic reticulum membranes were isolated from maturing endosperm tissue by sucrose density gradient centrifugation. The role of the endoplasmic reticulum (ER) in protein glycosylation was indicated by (a) localizing the enzymes catalysing the incorporation of N-acetylglucosamine and mannose into mono- and oligosaccharide lipid and into glycoprotein, (b) localizing particulate 3H-labelled glycoprotein amongst cellular fractions prepared from endosperm tissue which had been incubated with [3H]N-acetylglucosamine.

Glycoprotein fucosyl transferase in the endoplasmic reticulum of castor bean endosperm cells

Several proteins found in the major organelle fractions isolated from germinating castor bean endosperm are glycosylated [ 11. The analysis of sugars released from glycopeptides derived from organelle membrane and matrix subfractions indicated that fucose was associated with proteins located in the endoplasmic reticulum (ER) membrane [2]. Although L-fucose has been found in several plant glycoproteins (for example, lima bean lectin [3]), the glycosyltransferase which incorporates this sugar into glycoproteins has not been examined in plants [4]. The intracellular localization of glycoprotein fucosyltransferase in castor bean endosperm is of particular interest. Studies with mammalian tissues have established that whereas core sugars such as N-acetylglucosamine and mannose are added to nascent polypeptides by enzymes located in the rough ER membrane [S ,6] peripheral sugars such as fucose are added by enzymes located in the Golgi apparatus [7]. During the early post-germinative development of castor bean seedlings, the gluconeogenie endosperm cells do not divide [8 ] and the Golgi apparatus is not well developed in such cells [9,10]. The confinement of fucosylated glycoproteins to the ER membrane in castor bean endosperm [2] suggests that this fraction may be the major intracellular site of fucose incorporation. This has been confirmed in the present work.