Subcellular site of lectin synthesis in developing rice embryos

Abstract

Embryos of developing rice (Oryza sativa L. cv. Koshihikari) caryopses which actively synthesize lectin were labelled with [35S]cysteine for different times and newly synthesized rice lectin was isolated by affinity chromatography. Gel filtration of embryo extracts on Sepharose‐4B indicated that a large portion of the labelled lectin was associated with the particulate fraction. Experiments with detergent indicated that this lectin was sequestered within organelles. When extracts of pulse‐labelled embryos were fractionated on isopycnic sucrose gradients, this detergent‐released lectin banded in the same density‐region as the endoplasmic reticulum (ER) marker enzyme NADH‐cytochrome c reductase. Both radioactivity in rice lectin and the enzyme activity shifted towards a higher density in the presence of 2 mM Mg acetate, indicating that the labelled lectin was associated with the rough ER. The ER‐bound lectin could be chased from this organelle when tissue was incubated in unlabelled cysteine following a 1 h pulse of labelled cysteine. Radioactivity chased out of the ER with a half‐life of ˜4 h and accumulated in the soluble fraction. In the ER the lectin was present as a polypeptide with mol. wt. 23 000, while in the soluble fraction it occurred as polypeptides with mol. wt. 18 000, 10 000 and 8000. The rice lectin in the ER is capable of binding carbohydrates since it binds readily to the affinity gels. It is associated into dimers with an approximate mol. wt. of 46 000. The results show that newly synthesized rice lectin is transiently sequestered within the ER before further transport and processing take place.

Role of propeptide glycan in post-translational processing and transport of barley lectin to vacuoles in transgenic tobacco.

“…A total of 600,000 wt or gly-protoplasts were pooled and gently homogenized in 200 pL of 100 mM Tris, pH 7.8, 1 mM EDTA, 12% Sucrose (w/w) and separated into soluble and organelle fractions on Sepharose 48 columns (8.0 cm x 1.0 cm) according to Stinissen et al (1985). The Sepharose 4B elution profile of total radioactivity associated with organelles and soluble proteins concurred with previous studies (Stinissen et al, 1984(Stinissen et al, , 1985Mansfield et al, 1988). In addition, NADH-cytochrome-c reductase activity (Lord, 1983) was primarily associated with the organelle fractions.…”

Production of an Antibody Specific for the Propeptide of Wheat Germ Agglutinin

“…Triton X-00 was included to maximize the yield of organelle-associated lectin (11). The homogenate was centrifuged at l0,OOOg for 10 min to remove debris, and solid (NH4)2SO4 was added to 60% saturation.…”

Expression of rice lectin is governed by two temporally and spatially regulated mRNAs in developing embryos.

“…In the cultivated rice species Oryza sativa, the majority of the 18-kD subunits undergo a pro-' To whom correspondence should be addressed. teolytic cleavage event which yields two subunits of 8 kD and 10 kD (Stinissen, Peumans, and Chrispeels, 1984). This lectin is synthesized as a 23-kD monomeric precursor on the rough endoplasmic reticulum (RER) and is subsequently assembled into dimers within the lumen of the RER (Stinissen, Peumans, and Chrispeels, 1984).…”

“…teolytic cleavage event which yields two subunits of 8 kD and 10 kD (Stinissen, Peumans, and Chrispeels, 1984). This lectin is synthesized as a 23-kD monomeric precursor on the rough endoplasmic reticulum (RER) and is subsequently assembled into dimers within the lumen of the RER (Stinissen, Peumans, and Chrispeels, 1984). Assembled dimers are only transiently associated with the RER before being transported to and deposited in vacuoles/protein bodies (Stinissen, Peumans, and Chrispeels, 1984).…”

“…This lectin is synthesized as a 23-kD monomeric precursor on the rough endoplasmic reticulum (RER) and is subsequently assembled into dimers within the lumen of the RER (Stinissen, Peumans, and Chrispeels, 1984). Assembled dimers are only transiently associated with the RER before being transported to and deposited in vacuoles/protein bodies (Stinissen, Peumans, and Chrispeels, 1984). Rice lectin accumulates in specific cell layers of the scutellum, coleorhiza, radicle, root cap, and throughout cell layers of the coleoptile of embryos (Mishkind, Palevitz, and Raikhel, 1983).…”