A precursor of the reserve‐protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing <i>Phaseolus vulgaris</i> cotyledons

Bollini, Roberto; Wilden, Willem Van der; Chrispeels, Maarten J.

doi:10.1111/j.1399-3054.1982.tb02268.x

Cited by 48 publications

(28 citation statements)

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“…We have studied the intracellular trafficking of phaseolin, the major vacuolar storage glycoprotein of common bean seeds. In developing bean cotyledons, phaseolin is transported to the protein storage vacuoles (PSV): upon arrival at the PSV, a short propeptide is proteolytically removed from phaseolin (Bollini et al 1982, D'Amico et al 1992). This trimming event occurs at the C-terminus and involves only four or five amino acids (Bollini et al 1982, Bollini and Vitale, unpublished).…”

Section: Introductionmentioning

confidence: 99%

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

Frigerio

Foresti

Felipe

et al. 2001

Journal of Plant Physiology

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SummaryPhaseolin is a vacuolar storage glycoprotein synthesized as a precursor with a short C-terminal propeptide. We have recently shown that deletion of the last four C-terminal amino acids (AFVY, which are part of, or constitute the propeptide) abolishes vacuolar targeting, causing phaseolin to be secreted. Here we provide biochemical and microscopical evidence that the AFVY tetrapeptide, when fused to a secreted version of green fluorescent protein (GFP), inhibits GFP secretion and leads to its accumulation in vacuoles, where it is processed. This demonstrates that the tetrapeptide contains sufficient information for vacuolar sorting.

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Section: Introductionmentioning

confidence: 99%

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

Frigerio

Foresti

Felipe

et al. 2001

Journal of Plant Physiology

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“…In vivo labeling studies have also shown the appearance of 58 to 62 kD polypeptides after short-period labeling of oat caryopses (4). RER is thought to be the site of synthesis for legume reserve proteins (2). Recent evidence indicates that pea legumin is synthesized on the RER and transported into protein bodies, where it is processed into smaller acidic and basic subunits (6).…”

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Role of Endoplasmic Reticulum in Biosynthesis of Oat Globulin Precursors

Adeli¹,

Altosaar²

1983

Plant Physiol.

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Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin a-,8 dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced a-# dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the a + , subunits, were labeled in protein bodies.The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller a and ,B subunits.Oat seeds begin to synthesize a group of salt-soluble globulin polypeptides during development from 9 daf;2 thereafter, these globulins are predominant in the seeds among the four Osborne fractions (18,21). At maturity, the globulin fraction constitutes up to 75% of total oat protein in the groat (18,20). This is a unique characteristic of oat since other cereals such as wheat, barley, rye, and maize contain alcohol-soluble prolamins as the major storage protein fraction (14). Oat globulin is found in mature grain as a 12S multimeric molecule with an average mol wt of 348,000 (17). This molecule consists of 6 dimers, each dimer containing a 35 (a) and a 22 (13) kD subunit (17). The a and 1 subunits are apparently linked through disulfide bonds to form a dimer molecule with average mol wt 58,000 (3, 12, 21). Oat seed polysomes, when translated in vitro, give rise to two globulin polypeptides (60-62 kD) which are immunoprecipitable with globulin antibodies (4,12,23,26

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“…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.…”

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Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons

Chrispeels

Bollini²

1982

Plant Physiol.

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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 ...

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A precursor of the reserve‐protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Cited by 48 publications

References 37 publications

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

The C-terminal tetrapeptide of phaseolin is sufficient to target green fluorescent protein to the vacuole

Role of Endoplasmic Reticulum in Biosynthesis of Oat Globulin Precursors

Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons

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