Isolation of prolegumin from developing pea seeds: its binding to endomembranes and assembly into prolegumin hexamers in the protein storage vacuole

A fraction enriched in endoplasmic reticulum and Golgi membranesfrom developing cotyledons of Pisum sativum L. has proved tobe a convenient source for the isolation of prolegumin, theprecursor of the major 11S storage globulin of pea seeds. Twopro-proteins were isolated with molecular masses of 60 kDa and75 kDa, respectively. A monoclonal antibody, designated 2B1,against prolegumin was raised using the in vitro immunizationtechnique. This antibody recognizes the 60 kDa precursor polypeptide,but only the 20 kDa ß-subunit of mature legumin. Prolegumin,like the ß-subunit of the mature legumin, is a hydrophobicprotein. After import into the protein storage vacuole, andafter formation of the protein bodies trimeric 9S proleguminassembles into 12S hexamers without prior processing of theprecursor. Since prolegumin in vitro does not oligomerize intomore than 9S tnmers these results suggest that a protein-mediatedassembly of 9S prolegumin trimers into 12S prolegumin hexamersprobably occurs in the lumen of the protein storage vacuole.Prolegumin, but not mature legumin, binds very tightly to membranes.This property points to a possible way of identifying a putativeprolegumin receptor. Key words: Calcium, Endoplasmic reticulum, Golgi apparatus, legumim, monoclonal antibody, pea cotyledons     

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 (LeBDeltaN281) 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.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons

Cotyledons of developing pea seeds (pisum sativum L.) were labeled with radioactive amino acids and glucosamine, and extracts were prepared and separated into fractions rich in endoplasmic reticulum (ER) or protein bodies, The time-course of synthesis of the polypeptides of legumin and vicilin and the site of their assembly into protein oligomers were studied using immunoaffinity gels and sucrose density gradients. When cotyledons were pulse-labeled (1-2 h), newly synthesized vicilin was present as a series of polypeptides with M(r) 60,000-65,000, and newly synthesized vicilin was present as series of polypeptides with M(r) 75,000, 70,000, 50,000, and 49,000. These radioactive polypeptides were found primarily in the ER (Chrispeels et al., 1982, J Cell Biol., 93:5- 14). During a subsequent chase period, newly synthesized reserve proteins were initially present in the protein bodies in the above-named polypeptides. Between 1 and 20 h later, radioactive legumin subunits (M(r) 40,000 and 19,000) and smaller vicilin polypeptides (M(r) 34,000, 30,000, 25,000, 18,000, 14,000, 13,000, and 12,000) appeared in the protein bodies. The appearance of these labeled polypeptides in the protein bodies was not the result of a slow transport from the ER (or cytoplasm). Newly synthesized legumin and vicilin polypeptides were assembled into oligomers of 8S and 7S, respectively, in the ER. They appeared in the protein bodies in these oligomeric forms before the appearance of the smaller polypeptides (M(r) less than 49,000). These results indicate that the smaller vicilin polypeptides (M(r) less than 49,000) arise delayed posttranslational processing of some or all of the larger vicilin polypeptides. The precursors of legumin are completely processed in the protein bodies 2-3 h after their synthesis. The processing of the vicilin precursors is much slower (6-20 h) and only a fraction of the precursor molecules are processed. As a result both large (M(r) more than 49,000) and small polypeptides of vicilin accumulate in the protein bodies, whereas legumin accumulates only as polypeptides of M(r) 40,000 and 19,000.     

Role of the sulfhydryl redox state and disulfide bonds in processing and assembly of 11S seed globulins.

Seed legumins contain two conserved disulfide bonds: an interchain bond (IE) connecting the acidic and basic chains and an intrachain bond (IA) internal to the acidic chain. Mutant subunits were constructed in which these disulfide bonds were disrupted. Oxidized glutathione stimulated the rate of assembly of trimers with unmodified prolegumin subunits. Stimulation was not detected during assembly of IE mutant subunits and was diminished for the IA mutant. Hexamer assembly with trimers of mature unmodified subunits required oxidizing conditions. Trimers composed of mature IE mutants did not form hexamers. Both mutant and non-mutant subunits accumulated in hexamers when the cDNAs were expressed in tobacco. Hexamer assembly in seeds probably involved trimers with a mixture of mutant and non-mutant subunits. Similarly, mixed trimers that were a mixture of mutant and non-mutant subunits assembled into hexamers in vitro. The results demonstrate the importance of disulfide bonds during the assembly of 11S globulins.     

Characterisation of the storage protein subunits synthesised in vitro by polyribosomes and RNA from developing pea (Pisum sativum L.)

Evidence is presented to show that legumin, the major storage protein in Pisum, is synthesised in vitro by the wheat germ and reticulocyte lysate systems, from polyribosomes and mRNA isolated from developing pea seeds. While legumin isolated from mature pea seeds consists of 40,000 and 20,000 MW subunits, the in vitro legumin is synthesised as a 60,000 MW precursor consisting of covalently linked 40,000 and 20,000 MW subunits. The implications of these findings are discussed in relationship to studies with other systems.Abbreviations SDS-PAGE SDS-polyacrylamide gel electrophoresis - PBS phosphate buffered saline - MW molecular weight     

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.     

Different legumin protein domains act as vacuolar targeting signals.

Legumin subunits are synthesized as precursor polypeptides and are transported into protein storage vacuoles in field bean cotyledons. We expressed a legumin subunit in yeast and found that in these cells it is also transported into the vacuoles. To elucidate vacuolar targeting information, we constructed gene fusions of different legumin propolypeptide segments with either yeast invertase or chloramphenicol acetyltransferase as reporters for analysis in yeast or plant cells, respectively. In yeast, increasing the length of the amino-terminal segment increased the portion of invertase directed to the vacuole. Only the complete legumin alpha chain (281 amino acids) directed over 90% to the vacuole. A short carboxy-terminal legumin segment (76 amino acids) fused to the carboxy terminus of invertase also efficiently targeted this fusion product to yeast vacuoles. With amino-terminal legumin-chloramphenicol acetyltransferase fusions expressed in tobacco seeds, efficient vacuolar targeting was obtained only with the complete alpha chain. We conclude that legumin contains multiple targeting information, probably formed by higher structures of relatively long peptide sequences.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.     

Pulse-labeling Studies on Protein Synthesis in Developing Pea Seeds and Evidence of a Precursor Form of Legumin Small Subunit

Intact cotyledons were taken from pea seeds at various stages during seed development and pulse-labeled with ¹⁴C-amino acids. Salt-soluble proteins then were extracted and fractionated on Na dodecyl sulfate-polyacrylamide gels. Storage proteins in these extracts were identified by their binding to immunoaffinity columns. The labeling studies showed that the synthesis of storage protein polypeptides accounts for a major part of total protein synthesis of developing cotyledons between 10 and 22 days after flowering. The distribution of the incorporated radioactivity between individual storage protein polypeptides varied with stage of development. For example, the synthesis of the 50 kilodalton complex of vicilin subunits dominated the early stages of protein accumulation but was a negligible proportion of the total incorporation in the later stages. On the other hand, the 75 kilodalton vicilin subunit was synthesized throughout this entire period. The major small subunit of legumin (20 kilodaltons) was not detected by either Coomassie blue staining or by 2-hour labeling during this period. It was found to arise during the desiccation phase of seed maturation from a long-lived precursor with a relative electrophoretic mobility equivalent to 19 kilodaltons.     

Identification, Purification and Properties of the Precursor of Conglutin {beta}, The 7S Storage Globulin of Lupinus albus L. Seeds

Lupinus albus L. developing cotyledons 35 d after floweringcontained a major polypeptide of-average M_r 64000, immunologicallyrelated to conglutin ß, the 7S storage globulin ofthis seed. This polypeptide decreased during seed maturation,without completely disappearing in the mature seed. This dropwas accompanied by the formation of polypeptide fragments typicalof the mature conglutin ß. The 64000 polypeptide hasbeen identified as the precursor polypeptide of conglutin ß. Undenatured conglutin ß precursor, purified by ionexchange chromatography and size exclusion chromatography, showedsurface and association properties identical to the mature conglutinß molecule. The precursor oligomer, of M_r 190000,consisted of an association of three 64000 subunits. They stronglyreacted with concanavalin A indicating the presence of covalentlylinked carbohydrate. Tryptic treatment of the undenatured conglutin ß precursorled to the accumulation of a relatively stable 59000 polypeptidewhich was cleaved later on and produced three large polypeptidefragments differing from the mature conglutin ß polypeptides. Key words: Conglutin ß, precursor, developing seeds, Lupinus albus L.     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

In vitro proteolytic processing of pea and jack bean storage proteins by an endopeptidase from lupin seeds

The highly specific proteolytic breakdown observed upon prolonged treatment of pea legumin and pea and jack bean vicilin with a thiol endopeptidase purified from mature lupin seeds has been studied in detail. Proteolytic cleavage occurred in the acidic subunits of pea legumin, whereas the basic subunits were unaffected. Jack bean vicilin (M, 47 K) was cleaved near the middle of the polypeptide chain, whereas pea vicilin (M, 50 K) was cleaved into two fragments of M, 30 K and 20 K, respectively. The 30 K M, polypeptide chain contained covalently linked carbohydrate and had an N-terminal sequence suggesting that cleavage had taken place between the α and β region of the vicilin 50 K M, polypeptide as previously described in vivo. These results suggested that the cleavage specificity of lupin endopeptidase was in the proximity of paired arginine amino acid residues.The changes in the vicilin polypeptides due to proteolytic cleavage by lupin enzyme and those occurring during germination of pea seeds are also reported and discussed.     

Size and Charge Heterogeneity in Subunits of the Major Seed Storage Protein, Cucurbitin. Identification of a Polyprotein Precursor in Immature Seeds of the Water Melon Citrullus lanatus

Oil-rich seeds of the water melon (Citrullus lanatus (Thunb.) Mansf. ) also contain substantial amounts (30% w/w) of protein.The major storage globulin, called ‘ cucurbitin’,exists as a hexamer of non-covalently associated subunits ofMr 51000–54 000 which can be further dissociated in thepresence of disulphide-reducing agents to yield two groups ofpolypeptides of apparent Mr 33 000–37 000 and 20 000–25000 respectively. One- and two-dimensional polyacrylamide gelelectrophoretic separations on the basis of size and/or chargeconfirm that each polypeptide group is more heterogeneous thanpreviously suspected. A putative polyprotein precursor containingsequences related to each of the polypeptides of mature cucurbitincan be isolated and characterized from immature seeds. Cucurbitin is an important constituent of the diet in many Asianand African countries, a fact which prompted us to characterizemore fully the molecular structure of this valuable storageprotein as a prelude to future cloning and genetic manipulationexperiments. Key words: Seed storage protein, Precursor protein, Cucurbitin, Gel electrophoresis     

Storage protein accumulation in the absence of the vacuolar processing enzyme family of cysteine proteases

The role(s) of specific proteases in seed protein processing is only vaguely understood; indeed, the overall role of processing in stable protein deposition has been the subject of more speculation than direct investigation. Seed-type members of the vacuolar processing enzyme (VPE) family were hypothesized to perform a unique function in seed protein processing, but we demonstrated previously that Asn-specific protein processing in developing Arabidopsis seeds occurs independently of this VPE activity. Here, we describe the unexpected expression of vegetative-type VPEs in developing seeds and test the role(s) of all VPEs in seed storage protein accumulation by systematically stacking knockout mutant alleles of all four members (alphaVPE, betaVPE, gammaVPE, and deltaVPE) of the VPE gene family in Arabidopsis. The complete removal of VPE function in the alphavpe betavpe gammavpe deltavpe quadruple mutant resulted in a total shift of storage protein accumulation from wild-type processed polypeptides to a finite number of prominent alternatively processed polypeptides cleaved at sites other than the conserved Asn residues targeted by VPE. Although alternatively proteolyzed legumin-type globulin polypeptides largely accumulated as intrasubunit disulfide-linked polypeptides with apparent molecular masses similar to those of VPE-processed legumin polypeptides, they showed markedly altered solubility and protein assembly characteristics. Instead of forming 11S hexamers, alternatively processed legumin polypeptides were deposited primarily as 9S complexes. However, despite the impact on seed protein processing, plants devoid of all known functional VPE genes appeared unchanged with regard to protein content in mature seeds, relative mobilization rates of protein reserves during germination, and vegetative growth. These findings indicate that VPE-mediated Asn-specific proteolytic processing, and the physiochemical property changes attributed to this specific processing step, are not required for the successful deposition and mobilization of seed storage protein in the protein storage vacuoles of Arabidopsis seeds.     

Pea legumin overexpressed in wheat endosperm assembles into an ordered paracrystalline matrix

Legumin, a major component of pea seed storage vacuoles, is synthesized by a number of paralogous genes. The polypeptides are cleaved posttranslationally and can form mixed hexamers. This heterogeneity hampers structural studies, based on the production of hexamer crystals in vitro. To study a single type of homogenous legumin we produced pea legumin A in transgenic wheat (Triticum aestivum) endosperm where prolamins are predominant and only small amounts of globulins accumulate in separate inclusions. We demonstrated that the legumin precursor was cleaved posttranslationally and we confirmed assembly into 11S hexamers. Legumin was deposited within specific regions of the inclusion bodies. Angular legumin crystals extended from the inclusion bodies into the vacuole, correlating with the high legumin content. This suggests that the high-level production of a single type of legumin polypeptide resulted in the spontaneous formation of crystals in vivo. The use of a heterologous cereal system such as wheat endosperm to produce, isolate, and recrystallize homogenous 11S legume globulins offers exciting possibilities for structural analysis and characterization of these important seed storage proteins.     

The characterisation of vicilin during seed development in Vicia faba (L.)

Summary Vicilin and legumin were extracted from developing seeds at different stages using the classical method of repeated isoelectric precipitations. The subunits of these two protein fractions were separated by SDS gel electrophoresis, and it was shown that the sub-unit structure of vicilin changed during development whereas that of legumin did not. Thus vicilin is not a single protein.Vicilin was formed prior to legumin during seed development although the rate of synthesis of the latter was faster, so that in the mature seed the ratio of legumin to vicilin was about 4:1 by weight.     

Cell-free Synthesis of the Major Storage Protein of the Bean, Phaseolus vulgaris L

As seeds of the French bean (Phaseolus vulgaris, L. cv. Tendergreen) mature, a single protein, G1 globulin (analogous to legumin), represents the majority of protein synthesized. Washed polysomes extracted from developing cotyledons had little endogenous activity in amino acid incorporation, but on addition of cell-free extracts from wheat germ, active incorporation was obtained, the level being similar to that with viral RNA as messenger. The Mg(2+) optimum for protein synthesis in the presence of bean polysomes was 6 mm compared with 4 mm for synthesis of viral polypeptides in the wheat germ system. Using T-2 toxin as an inhibitor, it was shown that 29% of the incorporation depended on initiation events. Electrophoretic analysis of the total polypeptide products of cell-free synthesis gave a disperse profile. Centrifugation to remove polysome-bound peptides after 60 minutes incubation gave a supernatant having a product with the same electrophoretic mobility as G1 globulin and containing 26% of the radioactivity present in the gel. Protein eluted from this peak was subjected to re-electrophoresis and shown to consist of the three polypeptide subunits characteristic of G1 globulin.