Ronald Bassüner

The Role of Proteolysis in the Processing and Assembly of 11S Seed Globulins

11S seed storage proteins are synthesized as precursors that are cleaved post-translationally in storage vacuoles by an asparaginyl endopeptidase. To study the specificity of the reaction catalyzed by this asparaginyl endopeptidase, we prepared a series of octapeptides and mutant legumin B and G4 glycinin subunits. These contained amino acid mutations in the region surrounding the cleavage site. The endopeptidase had an absolute specificity for Asn on the N-terminal side of the severed peptide bond but exhibited little specificity for amino acids on the C-terminal side. The ability of unmodified and modified subunits to assemble into hexamers after post-translational modification was evaluated. Cleavage of subunits in trimers is required for hexamer assembly in vitro. Products from a mutant gene encoding a noncleavable prolegumin subunit (LeBΔN281) accumulated as trimers in seed of transgenic tobacco, but products from the unmodified prolegumin B gene accumulated as hexamers. Therefore, the asparaginyl endopeptidase is required for hexamer assembly.

Abundant embryonic mRNA in field bean (Vicia faba L.) codes for a new class of seed proteins: cDNA cloning and characterization of the primary translation product.

AbstractcDNA clones have been constructed bearing inserts for a specific mRNA class of high abundance in developing seeds of field bean (Vicia faba L.). Three full-length clones representing transcripts of different genes were sequenced and conceptually translated into a M=30 000 primary gene product. The structural analysis of the derived amino acid sequence revealed distinct domains: (i) a cleavable signal peptide; (ii) a hydrophilic N-terminal stretch possessing two serine clusters; (iii) a valine cluster and a hydrophobic domain in the C-terminal part of the polypeptide. The amino acid sequence of the polypeptide does not show homology with other known proteins.The corresponding mRNA could be isolated using cDNA clones and was efficiently translated in various systems. In a cell-free system the presence of a functional signal peptide was shown, which interacts with the signal recognition particle resulting in a cotranslational translocation across the membrane of the endoplasmic reticulum. If synthesized in Xenopus oocytes the translation product of the mRNA was secreted out of the cell. Homologous mRNA was found to be present also in developing cotyledons of pea (Pisum sativum L.) and french bean (Phaseolus vulgaris L.).

Characteristics of two types of legumin genes in the field bean (Vicia faba L.var. minor) genome as revealed by cDNA analysis

Nucleotide sequence analysis of cDNA clones coding for field bean legumin precursor polypeptides revealed two different types, called A and B. Although homologous, both types differ in several sequence characteristics. Comparison with similar data from soybean and recent findings from pea leads to the following conclusions: (i) the two types of legumin genes described represent two subfamilies, A and B, which are probably of widespread occurrence; (ii) legumin genes or subunits can best be placed in either subfamily A or B by sequence homology, in addition B‐type subunits contain generally fewer (or none at all in V. faba) Met residues as compared to A‐type subunits; (iii) members of one subfamily from different species are more homologous than members of either subfamily within a species, therefore the two subfamilies must have arisen long before speciation of the genera Glycine, Pisum and Vicia; (iv) during speciation members of the B‐subfamily diverged significantly more than members of the A‐subfamily.

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane

Hybridization‐selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell‐free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium‐washed membranes (K‐RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K‐RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557–561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.

Analysis of in vivo and in vitro Globulin Formation during Cotyledon Development of Field Beans (V icia faba L. var. minor)

Summary Cotyledons of field beans synthesize and accumulate the storage globulins vicilin and legumin concomitantly with the rapid cell elongation growth of storage tissue during stage 2 of embryogenesis. Biosynthesis of vicilin and legumin starts simultaneously when the cotyledon length reaches 2-3 mm. Vicilin is more actively formed in the beginning whereas legumin synthesis becomes the predominant event of globulin accumulation towards the end of developmental stage 2. Some minor globulin components of unknown nature appear in the middle of the globulin deposition period but do not contribute significantly to the storage protein quantity finally contained in mature seeds. In vitro translation of total cellular as well as of polysomal poly(A)-containing RNA leads to the formation of primary globulin polypeptides with larger Mr than that of in vivo formed globulins from mature seeds. The Mr of the primary in vitro translation products of legumin mRNA are of approximately similar size as the Mr of the disulfide-linked alpha- and beta-chains of in vivo formed mature legumins, thus suggesting that in accordance with other legume 11S globulins the subunits are covalently linked in a legumin propolypeptide. Cell-free translation of similar quantities of total cellular poly(A)-containing RNA from different developmental stages in a standardized wheat germ system reflects changes in the pattern of vicilin and legumin formation which correspond to the in vivo experiments. Polysome translation leads to similar results. The availability of mRNA for the globulin synthesis at cytoplasmic membrane-bound polysomes preferentially determines the pattern of vicilin and legumin formation during cotyledon development.

Narbonin, a novel 2S protein from Vicia narbonensis L. seeds: cDNA, gene structure and developmentally regulated formation

AbstractcDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (Mr ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.

Regulation von Biosynthese und Akkumulation der Reserveproteine wahrend der Entwicklung pflanzlicher Samen1)

Summary Biosynthesis and accumulation of storage proteins are mainly restricted to the cell elongation period (developmental stage 2) of storage tissues like the endosperm of cereal grains or the legume cotyledons. The rate increase of storage protein formation that takes place at the beginning of the cell elongation period seems to be regulated at the transcription level, i.e. by a rise in mRNA synthesis. mRNA of cereal and legume storage proteins is polyadenylated at the 3’-terminus and wears cap-structures at the 5’-end. cDNA copies of storage protein mRNA were synthesized by reverse transcription and molecular cloning of the double-stranded cDNAs was performed with Zea mays L., Hordeum vulgare L., Phaseolus vulgaris L., Pisum sativum L., Glycine max L. Merr., and Vicia faba L. Cell-free translation experiments led to the discovery of precursor proteins of the different storage protein polypeptides from cereals, whereas the two different polypeptides of the legume 11 S glo-. bulin of soybean, peas and field beans seem to be translated from one mRNA with the formation of one polypeptide which represents the common precursor of both polypeptides. All storage protein polypeptides are formed at membrane bound polysomes. Transfer of nascent storage protein polypeptides was observed from the polysomes that are attached to the exterior surface of maize protein bodies directly into these storage organelles, whereas in legume cotyledons the protein bodies are free of membrane bound polysomes and the mechanism of polypeptide transfer from the intracellular site of synthesis into the storage organelles still remains to be resolved.

Enrichment of Legumin mRNA by Immunoadsorption of Membrane-bound Polysomes from Developing Field Bean Seeds (Vicia faba L. var. minor)

Summary Polysomes synthesizing legumin were isolated by immunoadsorption using anti-legumin IgG from rabbits and anti-rabbit IgG-PAB-cellulose. The poly(A)-containing RNA from immunoselected membrane-bound polysomes exhibits a M r of 0.76 × 10 6 similar to the M r of the predominant peak of poly(A)-containing RNA from total mb polysomes which in addition shows minor components of M r 0.82–0.83 × 10 6 and 0.53 × 10 6 on a sucrose density gradient. Two legumin precursor polypeptides of Mr 64,000 and 67,000 were obtained by cell-free translation of poly(A)-containing RNA from immunoselected mb polysomes in the wheat germ system. Densitometer traces of in vitro translation products directed by poly(A)-containing RNA from both total mb polysomes and the immunoselected mb polysomes led to the calculation of an enrichment of 60–70% for the legumin mRNA by immunoselection of polysomes. No higher degree of enrichment could be achieved since polysomal aggregation and unspecific adsorption of the storage globulins to the polysomes interfere with the immunoselection procedure.