Abstract
Initially membrane bound polysomes, microsomes and total cellular polyadenylated RNA were isolated from developing cotyledons of Vicia faba. The templates were translated in vitro employing the wheat germ system (polysomes and microsomes) or the rabbit reticulocyte system (poly-A RNA). The translation products were isolated with antibodies raised against vicilin and legumin and analyzed by SDS-polyacrylamide gel electrophoresis. Vicilin was synthesized as one major N-terminally extended precursor molecule, with a molecular weight of about 52,000. The precursor was vectorially discharged through the membrane of the endoplasmic reticulum in conjunction with removal of the N-terminal extension. Legumin isolated from mature Vicia faba seeds consists of two subunits which are linked by cystin bridges and have molecular weights of about 36,000 and 20,000, respectively. These two subunits are known to be processed from a precursor molecule of 60,000 molecular weight. Applying antibodies raised against a purified legumin fraction for immunoaffinity isolation of in vitro translation products from polysomal, microsomal and mRNA templates yielded several polypeptides. The largest four of the polypeptides occurred in pairs with molecular weights around 51,000 and 60,000, respectively. These and one lower molecular weight polypeptide were co-translationally transported into the lumen of the endoplasmic reticulum. Hybrid microsomes were reconstituted from Vicia faba stripped microsomes and initially membrane bound polysomes isolated from barley endosperm. These microsomes served as an active template for the in vitro synthesis of hordein—the storage protein of barley endosperm. Furthermore, hordein was vectorially discharged through the Vicia faba microsomal membranes.
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Abbreviations
- DOC:
-
sodium deoxycholate
- DTT:
-
dithiothreitol
- HEPES:
-
N-2-hydroxyethyl-piperazine-N′-2-ethane sulfonic acid
- HKM:
-
HEPES-K-Mg acetate buffer (see 2.1)
- IgG:
-
immunoglobulin G; mol. wt.—molecular weight
- SDS-PAGE:
-
Sodium dodecylsulfate polyacrylamide gel electrophoresis
- TCA:
-
trichloroacetic acid
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Weber, E., Ingversen, J., Manteuffel, R. et al. Transfer of in vitro synthesized Vicia faba globulins and barley prolamins across the endoplasmic reticulum membrane of Vicia faba. Carlsberg Res. Commun. 46, 383 (1981). https://doi.org/10.1007/BF02907960
DOI: https://doi.org/10.1007/BF02907960