We present evidence that protein bodies constitute the principal lytic compartment in storage parenchyma cells of mung bean cotyledons and propose that they play a role in cellular autophagy. We developed a method to isolate protein bodies by incubating tissue slices with cell wall-degrading enzymes and fractionating the cellular organelles on a Ficoll gradient. About 75-80% of the protein bodies present in the protoplasts were recovered intact in a band at the 5/25% Ficoll interface. This band contained a similar proportion of the cellular alpha-mannosidase, N-acetyl-beta-glucosaminidase, ribonuclease, acid phosphatase, phosphodiesterase, and phospholipase D. beta-Amylase was present in the cells but not in the protein bodies. Ultrastructural observations showed that on the 3rd day of seedling growth protein bodies contain small vesicles (0.3-1.0 mum) with a cytoplasmic content (ribosomes, membrane vesicles, mitochondria). Later in seedling growth these vesicles appeared empty. We believe that these are autophagic vesicles resulting from invaginations of the protein body membrane and that their cytoplasmic contents are digested by the acid hydrolases present in the protein bodies.

1982. A presursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons. -Physio!. Plant. 55: 82-92.Developing cotyledons of Fhaseolus vulgaris L. were labeled for 30 min with pH] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein ofF. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with pH] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm~^) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mAf MgCU indicated that the newly-synthesized phaseolin was associated with the rough ER, Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseoiin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with ["CJ amino acids and pH] glucosamine or with ['"C] amino acids and pH] mannose, the PH]/["C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin. indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differentia) mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.Additional key words -Protein (storage), protein bodies.

The leaves of Phaseolus vulgaris L. cv Greensleeves contain an endopeptidase with a pH optimum of 9.0 and an isoelectric point between 10.0 and 10.5. This endopeptidase is the only abundant Azocoll-digesting proteinase in the leaves. The activity of this enzyme is highest in immature leaves and declines as the leaf matures and senesces. Enzymically isolated protoplasts contain very little of this proteinase. The proteinase can be recovered readily from the extracellular fluid obtained by gentle centrifugation of leaf strips vacuum-infiltrated with a buffered solution. These experiments indicate that the Azocoll digesting proteinase is located in the periplasmic space and/or the cell wall.Our present view of the structure of the cell wall is one in which cellulose fibrils are embedded in a three-dimensional matrix of hemicelluloses, pectic polysaccharides, and glycoproteins with hydroxyproline-rich domains. 3 To whom reprint requests should be addressed at Department of Biology, C-0 16, UCSD, La Jolla, CA 92093. described (3).Enzyme Extraction. Immediately after the leaves were taken from the plants, they were weighed, cut into small pieces, rinsed with distilled H20 and cooled to OC. The leaves were then homogenized in 4 volumes of 50 mm K-phosphate (pH 7.4) containing 1% insoluble PVP (Sigma). Homogenization was for 3 min at 0C with a Polytron homogenizer (Kinematica, Luzern, Switzerland). The homogenate was strained through four layers of cheesecloth and centrifuged at 25,000g for 15 min. The supernatant was used as a source of enzymes.Column Chromatography. Extract from 10 g of leaves were loaded on a DEAE-cellulose (Whatman, DE 32) column (30 x 1 cm), equilibrated with 50 mm K-phosphate (pH 7.4). The column was washed with equilibration buffer till the nonabsorbed protein was rinsed from the column and then eluted with equilibration buffer containing 1.0 M NaCl. Fractions were collected and assayed for enzyme activity. Azocollase activity was not absorbed and was recovered in the void volume of the column. The azocollase containing fractions were pooled, dialyzed against 1 mm K-phosphate (pH 7.4), and subjected to preparative isoelectrofocusing using a L. K. B. (Bromma, Sweden) 8001 column with a 1% ampholine (L. K. B.) solution in the pH 3.5 to 10.0 range. The column was run at 1 w and 4C for 40 h. Protoplast Isolation. Leaves were cut in 4-mm2 pieces, vacuum infiltrated, and incubated in a solution of 0.37% Cellulysin (Calbiochem) and 0.1% polycillin in mannitol medium (0.6 M mannitol in 10 mM Mes-NaOH buffer, pH 5.5). After 16 h of incubation at 27C with gentle shaking, the macerated tissue was resuspended in this medium and carefully strained through two layers of cheesecloth. The filtrate was centrifuged at 22g for 20 min. The pellet, which contained the intact protoplasts, was resuspended in mannitol medium containing 25% Ficoll and overlayered with a solution composed of 15% Ficoll in mannitol medium. The gradient was centrifuged at 40g for 15 min and the protoplasts formed a layer on top of t...

The subcelular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organeUle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.The protein bodies of mung bean cotyledons are large (I to 10 ytm) spherical organelles consisting of an amorphous protein matrix surrounded by a limiting membrane. They contain the reserve proteins vicilin and legumin (8) as well as a lectin (13) Recent work shows that a second acid hydrolase, ribonuclease, is also synthesized de novo during seedling growth and also accumulates in the protein bodies (15). Although its function in protein bodies may at first be less obvious, we have recently presented evidence indicating that the acid hydrolases found in protein bodies may function in the autophagic digestion of cytoplasmic structures (21). Here, we present evidence that ribonuclease and vicilin-peptidohydrolase are associated with RER-derived vesicles at the time when these enzymes are being synthesized. We suggest that the ER functions in the transport of these enzymes to the protein bodies. MATERIALS AND METHODS Plant Material and Extraction. Mung bean ( Vigna radiata (L.)Wilczek, cv Berken) seeds were purchased from W. Atlee Burpee Co., Riverside, Calif., and planted in moist Vermiculite after a 24-h imbibition. were harvested at appropriate times and homogenized in 2-4 ml of homogenization buffer (50 mM Tris-HCI [pH 7.5] with 1.0 mm EDTA and either 0.1 or 3.0 mM MgCl2) containing 12% (w/w) sucrose. The homogenate was cleared by centrifugation at 5OOg for 5 min.Purification and Fractionation of Organelles. Organelles were separated from soluble protein by chromatography on a Sepharose 4B (Pharmacia) column (15 mm diameter x 20 cm long). The column was loaded with 3 ml homogenate (10-20 cotyledons) and eluted with the homogenization medium. Fractions of 1.5 ml were collected, and the two fractions containing the most light-sca...

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