In vitro translation systems were prepared with supernatant factors from wheat germ and 80S ribosomes from wheat germ, barley embryos, watermelon cotyledons, pea cotyledons, and castor bean endosperm. Ricin A-chain, which strongly inhibits protein synthesis by mammalian ribosomes, inhibited all ofthe plant ribosomal systems by 50% when present at 25-45 ,ug/ ml--23,000 times the concentration needed to inhibit mammalian systems. Ricinus communi agglutinin A-chain, a protein similar to ricin A-chain, inhibited translation by the plant systems 50% at concentrations 5-10 times those of the ricin A-chain. Ribosomes from castor bean endosperm, the source of ricin and the agglutinin, were just as susceptible to the inhibitors as were ribosomes from the other four plants. Compartmentation of the inhibitors within vacuoles derived from protein bodies of the endosperm appears to be responsible for protecting cytoplasmic protein synthesis during germination of castor beans.Ricin and Ricinus communis agglutinin (RCA) are two glycoproteins present in the protein bodies of the endosperm tissue of seeds of castor bean (Ricinus communis) (1). Ricin, a potent toxin for animal cells, is composed oftwo subunits. The B-chain interacts with cell surfaces, allowing the toxin to enter cells, whereas the A-chain acts on ribosomes, thus inhibiting protein synthesis (2). In cell-free translation systems, the A-chain alone has a greater effect on translation than does the complete ricin molecule (2). RCA is structurally similar to ricin, as determined by immunologic relatedness (3) and amino-terminal sequences (4). However, RCA is composed of two A-chains and two Bchains and is much less toxic than ricin (5). The RCA A-chain can inhibit cell-free protein synthesis (4, 5), but Cawley et aL (4) have noted that ricin A-chain is 2 to 5 times more effective than RCA A-chain.Although there is general agreement in the literature on the ability of ricin A-chain to inhibit in vitro synthesis by mammalian 80S ribosomes, the information available for plant systems is confined to conflicting results on wheat germ. Olsnes et aL (2) cited unpublished work that demonstrated that a cellfree system from wheat germ "was strongly inhibited." Later, Cawley et aL (6) found no effect when ricin A-chain was added at 50 kg/ml to a poly(U)-directed wheat germ system, whereas ricin A-chain at 10 ng/ml inhibited a similarly prepared rat liver system by 50%. Recently, Lugnier and Rether (7) have demonstrated that ricin at 24 ug/ml inhibited a poly(U)-directed wheat germ system by 65%.The purpose of the present investigation was to study the effects ofricin, RCA, and their A-chains on cell-free translation systems composed of soluble factors from wheat germ and 80S ribosomes from wheat germ, barley embryos, watermelon cotyledons, pea cotyledons, and castor bean endosperm-the source ofricin and RCA. The results suggest some general conclusions about the response of plant translation systems to the two lectins.MATERIALS AND METHODS Plant Materials. Castor bean...
During seed development, various storage proteins and hydrolases accumulate in specialized storage vacuoles, the protein bodies, via an elaborate intracellular transport system involving the rough endoplasmic reticulum, the Golgi apparatus, and transit vesicles. Clathrin-coated vesicles, similar to those which transport lysosomal proteins to lysosomes, an organelle analogous to the vacuole, in animal cells, could be involved in this intracellular transport mechanism. Clathrin-coated vesicles have been isolated from cotyledons of developing pea (Pisum sativum L.) seeds at the time of rapid protein accumulation and analyzed for the presence of protein body constituents. A 23,000 M, polypeptide, corresponding to pea lectin precursor, was found associated with the vesicles, as determined by immunoblotting. The lectin precursor was apparently sequestered within the vesicles, as the polypeptide was only susceptible to proteolysis if detergents were included in the digestion buffer. A number of glycosidase activities, including a-mannosidase, a-galactosidase, and f8-Nacetylhexosaminidase, were also associated with the vesicles. Thus, it appears that clathrin-coated vesicles are involved in the intracellular transport of storage proteins during seed development.
The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10-day seedlings that had been exposed to light from day 5. The embryonic axes of 2-day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture. matrix and the globulin proteins of the crystalloid. The lectins are components of the matrix proteins (44,46) and are the only glycoproteins detectable in the protein bodies (8,44,46 Endosperm tissue was extracted with 1 N NaOH, using 2 ml per endosperm, by grinding in a mortar and pestle with sand. The homogenate was centrifuged at 20,000g for 15 min. The supernatant was decanted through two layers of Miracloth and the pellet re-extracted with 1 N NaOH. One ml of extract was mixed with 1 ml of 1 N HCI and 1 ml of 20% (w/v) TCA and held on ice for 5 to 10 min. The precipitated protein was pelleted and the pellet dissolved in 0.5 N NaOH. An appropriate dilution was assayed for protein (18), using BSA as the standard. The protein content of other extracts was assayed in a similar manner.Quantification of the Lectin Levels. All operations were conducted at 4°C. Twenty seedlings of known age were harvested and their endosperms extracted with 25 to 40 ml 0.1 M Na phosphate (pH 7.2), 0.15 M NaCl, 3 mM NaN3 by grinding in a mortar and pestle with sand. The extract was centrifuged at 20,000g for 15 min, the supernatant decanted through two layers of Miracloth, and the pellet re-extracted. The combined supernatant was brought to 65% saturation with (NH4)2SO4. A crude lectin fraction precipitated and was collected by centrifuging at 20,000g for 15 min. The (NH4)2SO4 pellet was suspended in PBS; 10 mm Na phosphate (pH 7.2), 150 mM NaCl, 3 mM NaN3, dialyzed against PBS, and loaded onto a Sepharose 4B-200 (Sigma) column equilibrated with PBS. The column was washed with PBS to remove contaminating proteins. Ricin was eluted www.plantphysiol.org on April 4, 2019 -Published by Downloaded from
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