Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons

Abstract

Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with 3H-amino acids, and newly synthesized phytohemagglutinin (PHA) The existence of membrane-bound lectin in the organs ofhigher plants is still a matter of some controversy. Bowles et al. (6) reported that Triton X-100 extracts of membranes from leaves, shoots, and roots of soybean possess high hemagglutinating activity. Recently Pueppke et al. (12) re-examined the question and concluded that the only organelle fractions containing hemagglutinating activity were from cotyledons of genotypes which also contain non-organelle-bound soybean agglutinin. They found no membrane-associated lectin in other soybean organs. They suggested that the lectin associated with the membranous organelles of cotyledons was not a cytoplasmic contaminant, but cautioned that corroborating evidence is needed to unequivocally establish the existence of membrane-bound lectin.Seed lectins such as soybean agglutinin, castor bean agglutinin, pea lectin, and PHA2 have all been shown to occur in the protein bodies of the cotyledons, together with the reserve proteins (1,3,10,17,18). Reserve proteins are made on membrane-bound polysomes (4, 11) and sequestered in the ER (2, 5, 7) prior to transport to the protein bodies (7). If lectins have a similar site of synthesis (4, 13) and pathway of transport, the ER probably contains a pool of lectin molecules en route to the protein bodies. In this paper, we provide evidence that this is indeed the case for phytohemagglutinin, the major seed lectin of Phaseolus vulgaris.MATERIALS AND METHODS Materials. Seeds of Phaseolus vulgaris L. cv Greensleeves (Burpee Seed Co., Riverside, CA) were grown as described (4). Experiments were carried out with cotyledons weighing 200 to 275 mg, when the accumulation of phaseolin and PHA is rapid (4, 15). Organic chemicals were purchased from Sigma Chemical Co. unless otherwise indicated. 3H-Amino acids were purchased from New England Nuclear Co.Radioactive labeling was carried out with excised cotyledons as described (14). The labeled tissue was collected by cutting a thin slice from the cotyledons with a razor blade. The remainder of the cotyledon was discarded. Homogenization, the use of Sepharose 4B to separate organelles from soluble molecules, and the use of continuous and discontinuous sucrose gradients have all been described (7, 16). NADH-Cyt c reductase was assayed as described (4).Affinity Chromatography. The affinity chromatography procedure of Felsted et al. (9) was used to separate PHA from other cellular proteins. Porcine thyroglobulin was linked to cyanogen bromide-activated Sepharose (Pharmacia) according to the specifications of the manufacturer. The thyroglobulin-Sepharose was used as an affinity gel in 0.25-ml portions in small plastic columns. The columns were washed exhaustively with PBS (0.15 M NaCl in 10 mm K-phosphate, pH 7.4), then with PBS containing 1 M NaCl, and then with 2 ml 50 mm glycine-HCl, pH 3.0, containing 0.5 M NaCl. The ...

Correct Post‐Translational Modification and Stable Vacuolar Accumulation of Phytohemagglutinin Engineered to Contain Multiple Methionine Residues

“…These results show that the [L38M, F94M, I96M1PHA mutant was efficiently transported in the secretory system. The rate of transport of [L38M,F94M,196MlPHA in these cells is similar to the rate of transport in bean cotyledons (Chrispeels and Bollini, 1982).…”

Subcellular site of lectin synthesis in developing rice embryos

“…Sequestration into the lumen of the ER is followed by transport out of the ER with a half-life of -4 h. The kinetics of transport of rice lectin from the ER appear to be slower than those reported for legumin and vicilin in developing pea cotyledons (90 min) (Chrispeels et al, 1982a) and phytohemagglutinin in developing bean cotyledons (90 min) (Chrispeels and Bollini, 1982), but are of the same order of magnitude as those reported for a-amylase in barley aleurone layers (5 h) (Jones and Jacobsen, 1982), and are faster than those of pea lectin in pea cotyledons (6-8 h) (Higgins et al, 1983).…”

“…Experiments with peas, beans and castor beans indicate that in these seeds lectins are made on the rough endoplasmic reticulum (ER) as pre-pro-proteins which lose their signal sequence,when they are sequestered in the lumen of the ER. The proteins are then transported to the protein bodies where proteolytic processing occurs (Higgins et al, 1983;Chrispeels and Bollini, 1982;Roberts and Lord, 1981). In beans (Phaseolus vulgaris) this transport is mediated by the Golgi apparatus (Chrispeels, 1983).…”

Developmental changes in the bark lectin of Sophora japonica L.

“…We explain these changes by proposing that, in summer, new tissue, which contains no lectin, is added to the bark from the inside while the cells in the outermost part degrade lectin, so that the total amount of lectin decreases. Biochemical studies have demonstrated that these proteins are synthesized on ERattached ribosomes and sorted into the ER lumen (Bollini and Chrispeels 1979;Bollini et al 1982;Chrispeels and Bollini 1982;; H u r k m a n and Beevers 1982; Faye and Chrispeels 1987), and it may be assumed that the bark lectin is also synthesized on ER-attached ribosomes. Our immunocytochemical data support this interpretation.…”