The Endoplasmic Reticulum of Mung Bean Cotyledons

Abstract

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...

Delivery of a Secreted Soluble Protein to the Vacuole via a Membrane Anchor1

“…5). Similar results were obtained when the vesicles were treated with 0.03% Triton X-100, a concentration of detergent known to release soluble microsomal proteins (Kreibich et al, 1973;Van der Wilden et al, 1980). We interpret these data to mean that the microsomes contain a 90-kD species of invertase that has no catalytic activity.…”

“…The purity and integrity of the vacuoles were monitored microscopically. To compare invertase activity in protoplasts and in the vacuolar fraction, equal ␣-mannosidase activities, determined according to the method of Van der Wilden et al (1980), were subjected to PAGE and stained for invertase activity as described above.…”

Biosynthesis, primary structure and molecular cloning of snowdrop (Galanthus nivalis L.) lectin

“…The organelles were separated from the soluble proteins and small molecules on a Sepharose 4B column (5 x 30 cm) as described by Van der Wilden et al (1980). Fractions containing the organelles were pooled and brought to 0.5% Triton X-100 and 0.5 M ammonium sulphate.…”

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

“…The organelles were separated from the soluble proteins and small molecules on Sepharose 4B columns (1.7 x 12 cm) (Pharmacia, Uppsala, Sweden) as described by Van der Wilden et al (13). Briefly, homogenates made in homogenization medium A or B were applied to Sepharose 4B columns equilibrated with medium A or B, respectively.…”

“…Further parts of the flower, especially the ovary (E Van Damme, unpublished data). The GNA' is a tetrameric protein built up of four identical subunits of M, 13,000, which does not contain carbohydrate (12).…”