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

Correspondence to: Niels C. Nielsen, nnielsen{at}dept.agry.purdue.edu (E-mail), 765-494-6508 (fax).

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

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 (LeBN₂₈₁) 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Many seeds accumulate sizable reserves of proteins in storage vacuoles during seed development, and a number of these proteins undergo a proteolytic cleavage as they accumulate in the vacuoles. Examples include post-translational modifications of 7S and 11S storage globulins, lectins, and 2S storage albumins (Casey et al. 1986 ; Nielsen et al. 1997 ). For many of these post-translationally modified proteins, cleavage takes place at a peptide bond that follows an asparagine residue. In many 11S storage proteins of seed from a wide variety of plant species, a single well-conserved Asn-Gly peptide bond is the target for cleavage. This event results in the formation of acidic and basic polypeptides that are characteristic of mature subunits (Dickinson et al. 1989 ). In a number of other cases, post-translational modification causes activation of proteolytic activity of the target molecule in seed storage vacuoles.

Asparagine-dependent endopeptidases that perform the post-translational modification of 11S reserve proteins have been described only recently (Ishii 1994 ). They have been purified from developing seeds of castor bean (Hara-Nishimura et al. 1991 ) and soybean (Scott et al. 1992 ; Hara-Nishimura et al. 1995 ), from mature seeds of soybean (Muramatsu and Fukazawa 1993 ) and jack bean (Abe et al. 1993 ), and from germinating seeds of vetch (Becker et al. 1995 ). That the purified endopeptidases have the required specificity to perform asparagine-dependent reactions has been demonstrated with synthetic peptides (Hara-Nishimura et al. 1991 ; Scott et al. 1992 ), with soybean proglycinin subunits synthesized in vitro (Scott et al. 1992 ), with 11S proglobulins isolated from developing seed (Hara-Nishimura et al. 1995 ), and with recombinant proglycinin synthesized in bacteria (Muramatsu and Fukazawa 1993 ). Although these studies established a requirement for an Asn residue on the N-terminal side of the peptide bond that was cleaved, information about the specificity of the enzyme for the other amino acids residing around the cleavage site has not been investigated.

This report describes studies to characterize the substrate specificity of the asparaginyl endopeptidase from developing soybeans. The ability of the endopeptidase to cleave eight synthetic octapeptides and 24 different synthetic 11S proglobulins with mutations around the conserved Asn-Gly cleavage site was evaluated. Our results show that the endopeptidase has an absolute specificity for Asn on the N-terminal side of the cleavage site but exhibits little specificity for amino acid residues on the C-terminal side. The endopeptidase appears involved in degradation of misfolded or foreign proteins that enter the vacuole. Finally, data are reported that proglobulin trimers, rather than hexamers, accumulate in vivo if the precursors cannot be cleaved post-translationally. Therefore, the endopeptidase is required for 11S hexamer formation during protein deposition in storage vacuoles.

	RESULTS

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Treatment of Proglobulin Trimers with Asparaginyl Endopeptidase
The 11S storage proteins normally are isolated from legume seed as hexamers. These hexamers are formed via a multistep pathway. Each subunit that participates in hexamer assembly is a product of one of a small family of genes. Precursor subunits derived from these genes are synthesized on rough endoplasmic reticulum (ER), and after removal of a signal peptide, they assemble into trimers in the lumen of the ER. Once assembled, the trimers traverse the plant secretory system to the protein storage vacuoles. Their accumulation in storage vacuoles is accompanied by the post-translational cleavage of the proglobulin subunit at a conserved Asn-Gly peptide bond. Cleavage of the trimers is associated with the appearance of hexamers in the vacuoles.

An in vitro system was developed to study steps in the assembly pathway (Dickinson et al. 1987 ; Jung et al. 1997 ; Nam et al. 1997 ). In this system, proglobulin subunits synthesized at the direction of cDNA self-assemble into trimers equivalent to those formed in the ER of developing seed. Whereas trimers formed in vivo probably have a heterogeneous composition with a mixture of subunits from each member of the gene family, those formed in vitro can have either a heterogeneous or homogeneous organization, depending on the composition of the mixture from which they are formed.

Scott et al. 1992 purified an asparaginyl endopeptidase from developing soybean that performs the post-translational maturation of proglobulin subunits. They demonstrated that the endopeptidase correctly processed a prolegumin B (LeB) from field bean and proglycinin-4 (G4) from soybean. It remains to be determined, however, whether the monomer or trimer is the preferred substrate of the endopeptidase.

To address this question, LeB was synthesized in vitro and permitted to self-assemble into trimers. The monomers that failed to assemble were then separated from trimers in sucrose density gradients. As shown in Figure 1, 40-kD acidic and 20-kD basic chains characteristic of the mature two-chain disulfide bonded subunits were recovered when LeB trimers were incubated with the asparaginyl endopeptidase (Figure 1, lanes 5 and 6). Once the acidic and basic chains from LeB trimers are formed, they are not degraded by continued exposure to the asparaginyl endopeptidase. In contrast, monomers from the sucrose gradients degrade completely when treated in the same manner (data not shown). It is possible, however, to observe transient cleavage products when samples are taken early during the digestion reaction (Figure 1, lanes 3 and 4). Each of the transient peptide fragments seen in Figure 1, lane 4, had a lower apparent molecular size than did the basic chain from mature subunits (Figure 1, lane 6). These results demonstrate that the monomers are unstable in the presence of the endopeptidase, presumably because they contain asparagine residues that are exposed to the endopeptidase. Apparently, such residues become inaccessible once trimers are formed.

View larger version (53K): [in this window] [in a new window]   Figure 1. Digestion of 11S Proglobulins with Asparaginyl Endopeptidase. Radiolabeled prolegumin B (pro LeB) subunits were synthesized and assembled in a rabbit reticulocyte lysate, as described in Methods. Purified and concentrated prolegumin trimers from sucrose gradient fractions were treated with asparaginyl endopeptidase from developing soybean cotyledons. The digestion products were separated by SDS-PAGE under reducing conditions and visualized by fluorography. Lanes 1 and 2, digestion of prolegumin B subunits permitted to assemble for 2 or 20 hr, respectively. In vitro translation was stopped by the addition of EDTA, and assembly occurred before aliquots were taken after either 2 or 20 hr of incubation. The samples were then digested with asparaginyl endopeptidase. Lanes 3 and 4, cleavage of purified monomeric prolegumins with heat-inactivated and active asparaginyl endopeptidase, respectively; lanes 5 and 6, purified prolegumin B trimers treated with heat-inactivated and active asparaginyl endopeptidase, respectively. Size markers (kD x 1000) are given at left.

Nam et al. 1997 demonstrated that monomers that fail to participate in self-assembly of trimers must be different from those that participate in trimer formation. This is because monomers that fail to incorporate into trimers immediately after they are synthesized are incapable of trimer formation later, despite their purification and reintroduction into conditions that should permit trimer assembly. We believe these monomers are misfolded. This raises the possibility that an assembly-competent monomer could exhibit a different sensitivity to digestion by the endopeptidase than those that are unable to participate in trimer assembly. Therefore, the sensitivity of the proteins in the mixture to the endopeptidase was compared after 2 and 20 hr of self-assembly. A comparison of lanes 1 and 2 in Figure 1 reveals that several digestion products besides the 40-kD acidic and 20-kD basic chains are visible after 2 hr that are not visible after 20 hr of assembly. These results, together with those obtained using monomers derived from the sucrose gradients, argue that trimers rather than monomers are the substrate encountered by the endopeptidase in protein storage vacuoles.

The time course for digestion of radiolabeled prolegumin and proglycinin by the endopeptidase was investigated. Figure 2A (left) compares the reaction products of radiolabeled LeB trimers after 20 and 90 min of digestion by the endopeptidase. Although the acidic and basic chains were the only products visible after 90 min, numerous transient reaction intermediates derived from the trimers were present 20 min after exposure to the endopeptidase. It is interesting that the acidic and basic chains of legumin were not the most prominent bands visible at 20 min.

View larger version (43K): [in this window] [in a new window]   Figure 2. Processing of Unmodified and High-Methionine Mutant 11S Proglobulins with Asparaginyl Endopeptidase. Purified radiolabeled trimers of the unmodified and mutant proglobulin subunits were treated with asparaginyl endopeptidase. The products were separated by SDS-PAGE 20 and 90 min after the initiation of digestion. In addition to acidic and basic chains as the main cleavage products, digestion of proglycinin G4 gives rise to c1 and c2 fragments due to the presence of the secondary cleavage site at Asn55 in the acidic chain. Treatment of the proglycinin G4RM5 mutant with asparaginyl endopeptidase virtually results in a complete degradation, leaving no acidic or basic chains after cleavage. Size markers (kD x 1000) are given at left. (A) Digestion of prolegumin B (pro LeB) and its methionine-enriched mutant LeB-Met. (B) Digestion of proglycinin G4 (pro G4) and its methionine-enriched mutant G4RM5.

An equivalent picture emerged when this experiment was repeated with G4 proglycinin trimers (Figure 2B, left), although interpretation of these data is complicated by the fact that the G4 proglobulin subunit contains two potential cleavage sites. One is the normal cleavage site after Asn₃₅₅, and the other is after Asn₅₅ (Staswick 1982 ; Staswick and Nielsen 1983 ; Kitamura et al. 1984 ). Whereas cleavage at Asn₃₅₅ leads to the formation of the acidic and basic chains, cleavage after Asn₅₅ results in two fragments from the acidic chain. The larger fragment (c1) originates from the C-terminal part of the acidic chain, and the smaller fragment (c2) comes from the N-terminal part. Although fewer transient digestion fragments are recovered from G4 compared with LeB, transient reaction intermediates from G4 are visible after 20 min that are not evident 90 min after endopeptidase treatment. Fewer transient digestion intermediates from G4 could reflect a rapid degradation of the fragments by the endopeptidase or could be the consequence of fewer reactive Asn residues in G4 compared with LeB.

We interpret the appearance of transient digestion intermediates to indicate that there are at least two types of proglobulin subunits in the purified trimer preparations. One type of proglobulin subunit is folded correctly, whereas the other type is misfolded. The latter, although misfolded, is apparently still able to participate in trimer assembly. When exposed to the endopeptidase, those subunits that are misfolded degrade rapidly, whereas those that are folded correctly are slowly processed into the mature two-chain subunits.

Several mutant G4 subunits have been produced that contain additional methionine residues in their hypervariable region (Dickinson et al. 1987 , Dickinson et al. 1990 ; Lago et al. 1991 ). G4RM5 and G4RM6, which contain five and six Arg-Met residues, respectively, are examples of such mutants (Dickinson et al. 1990 ). Saalbach et al. 1988 also described a mutant (LeB-Met) that contains additional methionine residues in its basic chain. We asked how trimers assembled from these mutant subunits would respond to treatment with the asparaginyl endopeptidase. The right panels in Figure 2A and Figure 2B, respectively, present results obtained with trimers of mutant LeB-Met and G4RM5 subunits. In each case, the trimers were degraded completely by the endopeptidase. Similar results were obtained with G4RM6 and the other additional high-methionine G4 glycinin mutants that were described by Lago et al. 1991 (data not shown).

To determine whether mutant G4RM5 and G4RM6 subunits were accumulated in developing seed, cDNAs encoding the mutants were placed under the control of a glycinin-4 gene promoter, introduced into pBIN19, and transformed into tobacco. Protein extracts of seed from transformed plants were separated by SDS-PAGE and analyzed in protein blots by using an antibody against glycinin that preferentially recognized acidic chains. Even though ELISA signals to glycinin could be detected in extracts from seed that contained the mutant glycinin genes (Lago et al. 1991 ), the data in Figure 3 show that intact acidic chains were not detectable when protein blots of these extracts were probed with antiglycinin immunoglobulins. Importantly, signals due to acidic chains were present in seed extracts from control plants that harbored unmodified G4 coding sequences. Because proteases besides the asparaginyl endopeptidase are present in seed storage vacuoles, one cannot conclude that only the asparaginyl endopeptidase was responsible for the instability of G4RM5 and G4RM6 in recombinant tobacco. However, it is clear that the asparaginyl endopeptidase may contribute to their instability.

View larger version (36K): [in this window] [in a new window]   Figure 3. Analysis of G4RM5 and G4RM6 Subunit Accumulation in Transgenic Tobacco. Seed extracts from transgenic tobacco were separated by SDS-PAGE under reducing conditions, blotted onto nitrocellulose, and analyzed using an antibody that interacts preferentially with acidic glycinin chains. Size markers (kD x 1000) are given at left, and the position to which the glycinin acidic chains migrate is shown at right (arrowhead). Lanes containing purified glycinin were included on both sides of lanes, with tobacco seed extracts as controls. As indicated in lanes with untransformed tobacco extracts, a number of proteins cross-react with the antibody. However, tobacco line G4, which is transformed with the nonmodified glycinin-4 gene (Gy4), accumulates a protein the same size as the controls. Intact acidic chains derived from mutants G4RM5 and G4RM6 are not detectable.

Substrate Specificity of Asparaginyl Endopeptidase as Determined with Octapeptides
A series of mutant peptides and 11S proglobulin subunits was constructed to study substrate specificity of the asparaginyl endopeptidase. Consistent with practices customarily used to describe the specificity of many other proteases, we use the nomenclature by Schecter and Berger 1967 to identify the amino acid residues around the peptide bond cleaved by the enzyme. Consistent with this nomenclature, and as indicated in Figure 4A, the amino acids that flank the cleaved peptide bond are P1 on the N-terminal side and P1' on the C-terminal side. The residues are numbered consecutively in either direction from the cleavage site.

View larger version (64K): [in this window] [in a new window]   Figure 4. Amino Acid Sequences of 11S Proglobulins and the Cleavage Site Mutants around the Site Where Post-Translational Processing by the Asparaginyl Endopeptidase Takes Place. (A) Comparison of sequences of LeB and G4 with a consensus sequence of legume 11S globulin around the conserved processing site. Although a few amino acids, including P1 Asn and P1' Gly, are highly conserved, other residues toward either the N-terminal (left) or C-terminal end (right) of a globulin molecule display varying degrees of divergence. Cleavage between P1 and P1' residues by the endopeptidase gives rise to the acidic and basic chains that comprise mature subunits. Amino acid color code: green, basic; red, acidic; blue, polar; white, neutral; yellow, hydrophobic. (B) Sequences and cleavage by asparaginyl endopeptidase of various synthetic octapeptides containing substitutions for cleavage site amino acids. The unmodified sequences are represented by a peptide containing the amino acids found at the processing site of proglycinin G4 (G4 352 to 359). Mutants are denoted by an abbreviation for the mutant amino acid(s) following the position of modification(s). For example, G4Q355 represents a change from N355 to Q355 at position P1 of the cleavage site. The synthesized peptides were subjected to digestion by the endopeptidase, and the products were analyzed by thin-layer chromatography (Scott et al. 1992 ). (C) Sequences and cleavage by asparaginyl endopeptidase of LeB and its mutants. The various mutants of LeB were constructed by site-directed mutagenesis. In vitro–synthesized and labeled trimers of the unmodified and mutant subunits were subjected to digestion by the endopeptidase. Results from the cleavage reactions as analyzed by SDS-PAGE and fluorography are summarized. The acidic and basic polypeptides derived from the cleavage of unmodified subunits are represented by bars. A in names of mutants indicates a deletion. Figure 4. (continued). (D) Sequences and cleavage by asparaginyl endopeptidase of proglycinin G4 and its mutants. Proglycinin G4 contains a secondary cleavage site at Asn55, which results in the production of c1 and c2 after proteolytic cleavage. In addition to the mutants with amino acid substitutions introduced around the conserved processing site at Asn355, a secondary cleavage site mutant (G4Q55) was also constructed. Results from the digestion of trimers from these mutants and unmodified G4 are shown. In general, mutations that change the P1 Asn residue cause a loss of cleavage. With several exceptions, mutations at positions other than P1 do not prevent cleavage, and the products remain stable in the presence of the endopeptidase even after prolonged incubations. Mutants LeBR282F283 and G4Q357 are exceptions (denoted by an asterisk). In these, the mutations destabilize the subunits and cause degradation. Mutant LeBI282G283 was not cut by the endopeptidase. aa, amino acid.

The 13 amino acid residues that span the conserved post-translational cleavage site in both G4 and LeB are displayed in Figure 4A. As summarized in Figure 4A, several interesting features emerge when sequences from legume 11S subunits are compared. First, an asparagine residue at position P1 is conserved in all of these proteins. Second, glycine seems to be the preferred residue at the P1' position among the legume proteins, although other amino acids occur at this position in some gymnosperms (Nielsen et al. 1997 ). The amino acids on the C-terminal side of the cleaved peptide bond appear to be more highly conserved among divergent 11S sequences than are the residues on the N-terminal side. Where variability is encountered on the P' side of the cleavage site, the changes are conservative in the sense that the chemical properties of the normal and substituted amino acids are preserved. Finally, the disulfide bond that links the acidic and basic peptides in the mature subunit requires the cystine found at position P7' (Staswick et al. 1984 ).

To study the substrate specificity of the asparaginyl endopeptidase toward amino acids around the conserved Asn-Gly peptide bond in 11S storage proteins, the series of octapeptides described in Figure 4B was synthesized. The octapeptide ETRNGVEE corresponds to amino acids 352 to 359 in proglycinin G4 (Scallon et al. 1987 ) and is cleaved by the endopeptidase purified from seed, as reported earlier by Scott et al. 1992 . Replacement of the asparagine at position P1 with glutamine, aspartate, alanine, isoleucine, or valine resulted in a loss of cleavage when the mutant octapeptides were challenged by the asparaginyl endopeptidase. Each of the octapeptides with mutations at position P1 remained stable in the presence of the endopeptidase, even after prolonged periods of incubation. Replacement of P1' glycine with glutamate, or replacement of P1'-P2' Gly-Val with Ile-Gly, did not prevent the endopeptidase from cutting the octapeptide. These results show that the endopeptidase requires Asn at P1, but there is less specificity toward amino acids on the C-terminal side of the peptide bond that is cleaved.

Cleavage of Mutant 11S Proglobulin Subunits with the Asparaginyl Endopeptidase
The specificity of the seed asparaginyl endopeptidase was also evaluated using a series of LeB subunits that contained mutations at every position between P6 and P7' (Figure 4C). For these experiments, cDNAs that encoded prolegumin B were modified by site-directed mutagenesis as described by Jung et al. 1992 . Radioactive mutant subunits were synthesized using the cDNAs (Dickinson et al. 1987 ), self-assembled into 9S trimers, and purified in sucrose density gradients. The trimers recovered from the gradients are composed principally of intact proglobulin subunits (Dickinson et al. 1990 ).

The trimers made with each of the LeB mutant subunits were challenged by the asparaginyl endopeptidase. Results of these experiments are summarized in Figure 4C, and representative examples of the experiments are shown in Figure 5. As is the case with LeBP₂₇₉ and LeBA₂₇₄ (Figure 5A), treatment of most mutants resulted in the appearance of acidic and basic chains. Once formed, the chains remained stable in the presence of the endopeptidase even after prolonged incubation (data not shown). Two of the modified subunits contained mutations at position P1. In one, LeBQ₂₈₁, a Gln replaced Asn₂₈₁. In the other, LeBN₂₈₁, Asn₂₈₁ was eliminated. Like LeBQ₂₈₁ (Figure 5B), LeBN₂₈₁ was not cut by the endopeptidase, even after prolonged incubation. A few mutants, such as LeBR₂₈₂F₂₈₃, presented in a special way because acidic and basic chains from these mutants could not be detected among the cleavage products (Figure 5B). As the cocleavage experiments described below show, the precursors and products were completely degraded by the endopeptidase.

View larger version (41K): [in this window] [in a new window]   Figure 5. Processing of LeB and Proglycinin G4 Cleavage Site Mutant Subunits by the Asparaginyl Endopeptidase. The mutant subunits were synthesized in vitro and self-assembled into trimers. Purified trimers from sucrose gradients were concentrated before being subjected to cleavage by the endopeptidase. The digestion was performed at 30°C for 90 min with either heat-inactivated (lanes 1 in [A] to [C]) or active endopeptidase (lanes 2 in [A] to [C]; lanes 1 in [D]), respectively. As controls, the unmodified and mutant subunits were mixed 1:1 (hydrogen-3 counts per min) and digested with active endopeptidase (lanes 3 in [A] to [C]; lanes 2 in [D]). In (C), proglycinin secondary cleavage site mutant G4Q55 was digested with active endopeptidase (lane 4) next to the unmodified G4 trimers (lane 5). In each case, the mutant and unmodified trimers were differentially labeled to facilitate calculation of the relative digestion of each type of trimer (see Methods). Products from the cleavage reactions were analyzed by SDS-PAGE and fluorography. (A) Cleavage of prolegumin B mutants LeBP279 and LeBA274. (B) Cleavage of prolegumin B mutants LeBQ281 and LeBR282F283. (C) Cleavage of proglycinin G4 mutants G4D355 and G4Q55. (D) Cleavage of proglycinin G4 mutants G4L356, G4S356, and G4Q357.

Analogous results were obtained when trimers made from mutant G4 proglycinin subunits were evaluated. The data in Figure 5C show that replacement of Asn₃₅₅ at P1 with an Asp produced a mutant proglycinin subunit that could not be cleaved even after prolonged incubation with the endopeptidase. As discussed earlier, G4 is unique among the proglycinins because its acidic chain contains a secondary cleavage site after Asn₅₅. Replacement of Asn₅₅ with Gln (i.e., mutant G4Q₅₅) eliminated cleavage of the subunit at the secondary site (Figure 5C), indicating that the presence of the mutation prevented formation of the c1 and c2 fragments. On the other hand, and consistent with the behavior of corresponding LeB mutants, the replacement of Gly₃₅₆ in the P1' position with either a Leu or Ser did not prohibit cleavage (Figure 5D). In the case of the G4Q₃₅₇ mutant, however, the replacement of Val₃₅₇ at P2' with Gln caused the endopeptidase to degrade rapidly and completely the precursor-like LeBR₂₈₂F₂₈₃. Taken together, the results summarized in Figure 4 and Figure 5 showed that the vacuolar asparaginyl endopeptidase requires Asn at position P1 but is far less dependent on the other amino acids that surround the cleavage site.

Kinetics of Digestion by Asparaginyl Endopeptidase
A cocleavage assay was developed to determine whether mutations around the conserved Asn-Gly cleavage site altered the kinetics of the reaction catalyzed by the asparaginyl endopeptidase. All of the mutants described in Figure 4 were tested, but only examples representative of the results obtained are reproduced in Figure 6. In these experiments, trimers consisting of mutant or unmodified subunits were each labeled with a different radioactive nuclide to facilitate their detection and quantification. The differentially radioactively labeled trimers were mixed and treated with the endopeptidase; then the digestion products were separated by SDS-PAGE. Radioactivity in fractions recovered from the gel was determined and used to calculate the relative amount of digestion of the control and mutant subunits in the manner described in Methods. Results after the proteolysis of modified subunits are shown at right in Figure 6, whereas results at left show the behavior of internal controls.

View larger version (36K): [in this window] [in a new window]   Figure 6. Cocleavage Analysis of Wild-Type and Mutant 11S Proglobulins by Asparaginyl Endopeptidase. Purified 14C-leucine–labeled mutant proglobulin trimers and 3H-leucine–labeled wild-type proglobulin trimers were combined 1:1 in volume and digested with asparaginyl endopeptidase at 30°C, pH 6. At 10, 50, and 90 min after the initiation of digestion, aliquots were taken and separated by SDS-PAGE. Bands that appeared on the fluorograph were sliced out of the gel and immersed in scintillation cocktail, and their radioactivity was determined by scintillation counting. The counting results were used to calculate the quantities of hydrogen-3 and carbon-14 labels, each associated with either the precursor protein or the acidic/basic chain, as described in Methods. The time course of cleavage as percentage of radiolabels associated with each peptide band is shown. (A) Cocleavage of wild-type G4 and its mutant protein G4L356. (B) Cocleavage of wild-type G4 and its mutant protein G4D355. (C) Cocleavage of wild-type G4 and its mutant protein G4Q357.

As is evident from the data in Figure 6, a two-phase reaction was observed for each of the samples. During the first phase, there was a rapid disappearance of both the mutant and unmodified subunits, and an accumulation of acidic and basic chains that corresponded with the disappearance of the proglobulin did not take place during this initial reaction phase. For most of the mutants, the acidic and basic chains appeared during the second, more linear, reaction phase. We believe that the first reaction phase reflects a digestion of subunits that are folded incorrectly but are still capable of assembly into trimers. Accordingly, the second phase of the reaction reflects the cleavage of properly folded subunits.

Three types of kinetic behavior during the second reaction phase were identified when trimers of the mutant subunits were treated with the endopeptidase. In the most common form, the mutant subunits seemed to be processed at rates that were similar to those observed for trimers composed of unmodified prolegumin. The course of the reaction shown for mutant G4L₃₅₆ is typical for this group (Figure 6A). The acidic and basic polypeptides appeared as the proglobulins disappeared. And the timing of their appearance was the same as when unmodified subunits were processed. For mutants with the second type of kinetic behavior, there was no response to the endopeptidase during the second phase of the reaction. This group included mutants in which the Asn at the P1 position was changed (Figure 6B), and the cleavage of G4D₃₅₅ is characteristic for them. This result was anticipated because correctly folded G4D₃₅₅ subunits could not be processed by the endopeptidase. Curiously, trimers formed from LeBI₂₈₂G₂₈₃ subunits were like G4D₃₅₅, LeBN₂₈₁, and LeBQ₂₈₁ in that they were not cleaved (Figure 4C). The third type of kinetic behavior is exemplified by mutant G4Q₃₅₇ (Figure 6C). For this group of mutants, cleavage by the endopeptidase resulted in complete and rapid degradation of the proglobulins. It is likely that the amino acid changes in these mutants resulted in structural changes that exposed asparagine residues and facilitated subunit digestion.

Cleavage of Proglobulins Promotes the Assembly of Trimers into Hexamers
The capacity of the asparaginyl endopeptidase to promote assembly of trimers into hexamers was evaluated. For this experiment, radiolabeled prolegumin B trimers were synthesized and assembled in vitro (Dickinson et al. 1987 ). The trimers that resulted were treated with either an active or heat-inactivated asparaginyl endopeptidase preparation. The reaction mixtures were supplemented with nonradioactive subunits of dissociated mature soybean glycinin at an ionic strength appropriate to induce reassociation into hexamers (Dickinson et al. 1989 ). After separation of the reassembly products in sucrose density gradients, samples from the gradient were analyzed for radioactivity. The data in Figure 7A show that radioactivity became associated with the hexamer fraction only when the prolegumin B trimers were treated with the active endopeptidase. Trimer samples treated with heat-inactivated enzyme failed to incorporate into hexamers. The 9S trimer and 11S hexamer fractions from the gradient were analyzed by SDS-PAGE and followed by fluorography to identify the positions of radiolabeled proteins (Figure 7B). The 11S fraction was composed exclusively of mature cleaved subunits, whereas the 9S fraction contained both processed and unprocessed proteins. These results demonstrate that cleavage of the proglobulins in trimers is associated with the formation of 11S hexamers in vitro.

View larger version (23K): [in this window] [in a new window]   Figure 7. Reassembly of Prolegumin B Processed by Asparaginyl Endopeptidase. (A) Sucrose density gradient separation of processed and reassembled prolegumin B. Purified 3H-leucine–labeled prolegumin B trimers were digested in 0.02 M citrate-phosphate buffer, pH 6.0, for 3 hr in the presence of either heat-inactivated (open circles) or active (solid circles) asparaginyl endopeptidase. After digestion, mixtures were adjusted to pH 7.0, and dissociated glycinins were added to the final concentration of 1.25 mg/mL (Dickinson et al. 1989 ). Reassembly took place at 25°C for 15 hr, and products were separated by sucrose gradient centrifugation. One-third of each gradient fraction was assayed for radioactivity. (B) Sucrose gradient fractions corresponding to brackets (a to d) shown in (A) were pooled, trichloroacetic acid precipitated, and subjected to SDS-PAGE. Whereas part of the aliquots from the 9S peak (lane b) comigrated with proglobulin B (pro LeB) trimers, samples from the 11S peak (lane c) contained acidic and basic chains exclusively. Unseparated digestion samples from (A) are shown as controls in lanes 1 (heat-inactivated enzyme) and 2 (active enzyme).

Evidence was sought that the post-translational cleavage of subunits was required for the assembly of hexamers. These experiments relied on results described by Baumlein et al. 1987 . These investigators expressed a legumin B gene from field bean in tobacco and demonstrated that acidic and basic chains typical of mature 11S subunits were detected in seed from the transgenic plants. Because tobacco plants were capable of processing transformed prolegumin B correctly, the system was considered suitable to test cleavage site mutants in vivo. Accordingly, cDNAs encoding unmodified LeB and mutant LeBN₂₈₁ were ligated to the legumin B promoter and introduced into tobacco. DNA and RNA gel blot hybridization experiments were conducted to select transgenic tobacco plants expressing legumin mRNA from single-copy insertions (data not shown). To demonstrate that the proteins were targeted to protein storage vacuoles, anhydrous glycerol homogenates were prepared from tobacco seed that expressed the transgenic storage protein subunits and were separated in a potassium iodide–glycerol gradient (Sturm et al. 1987 ). The vacuolar marker enzyme -mannosidase cofractionated with the proglobulins from both LeB and the LeBN₂₈₁ mutant (data not shown). Therefore, the products of the transgenes were correctly targeted to storage vacuoles. The proteins of the storage vacuoles were resolved by SDS-PAGE either in the presence or absence of the sulfhydryl reductant 2-mercaptoethanol. As the data in Figure 8 show, when protein blots from these gels were probed with legumin B–specific antibodies, acidic chains were detected in extracts of tobacco transformed with the gene encoding unmodified LeB. However, seed extracts from transformants with the mutant LeBN₂₈₁ gene contained a legumin protein derivative that migrated to the same position as uncleaved prolegumin. Thus, an Asn in position P1 is required for cleavage of prolegumins in vivo.

View larger version (40K): [in this window] [in a new window]   Figure 8. Accumulation of Prolegumin B and Its Mutant Polypeptide LeBP1N281 in Transformed Tobacco Seed. Recombinant gene constructs coding for either wild-type prolegumin B or its mutant LeBP1N281 were transformed into tobacco. Seed extracts were prepared and resolved by SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane, and then the filter was hybridized with legumin B–specific polyclonal antibodies that interacted preferentially with acidic chains. Lane 1, untransformed tobacco seed extract (WT); lane 2, extract from transformant with mutant LeBP1N281 (N); lane 3, extract from transformant with the wild-type prolegumin B gene (LeB); lane 4, reduced field bean legumin control (R); and lane 5, unreduced field bean legumin control (NR). Numbers at left are molecular weights (MW). pro LeB, prolegumin B; Vf, Vicia faba (fava bean).

To characterize tobacco that expressed unmodified legumin B and mutant LeBN₂₈₁ genes, seed extracts were resolved by centrifugation in sucrose density gradients. Proteins from the gradient fractions were separated by SDS-PAGE and analyzed in protein blots probed with LeB-specific antibodies. The data in Figure 9a show that acidic and basic chains derived from the unmodified legumin B gene were recovered in the 11S fraction, whereas the data in Figure 9b show that prolegumins derived from LeBN₂₈₁ were found uncleaved in the 7S globulin fractions. These results reveal that the mutant prolegumin B subunits do not assemble into hexamers in vivo, a finding that demonstrates that cleavage of the precursor is required for assembly of proglycinin and prolegumin trimers into hexamers in vitro.

View larger version (103K): [in this window] [in a new window]   Figure 9. Accumulation of LeBN281 Mutant Subunits as Trimers in Transgenic Tobacco Seed. Extracts from transgenic tobacco seed expressing either the wild-type legumin B or its mutant LeBN281 protein were separated in sucrose density gradients. The samples from the fractions designated were separated by SDS-PAGE, proteins on the gel were blotted, and the filters were probed immunologically with anti–legumin B antibody preparations. Left lanes show controls not separated by centrifugation. (A) Separation of extract from a transformant with the wild-type prolegumin B gene (LeB). (B) Separation of extract from a transformant with the mutant LeBN281 gene (LeBN281).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The amino acid sequences are highly conserved around the Asn-Gly bonds in 11S storage globulins that are cleaved post-translationally. We considered the possibility that the high degree of structural conservation might reflect a restricted specificity of the asparaginyl endopeptidase responsible for this cleavage. However, when various octapeptides and mutant prolegumins and proglycinins with mutations in this conserved region were challenged by endopeptidase, it became clear that this was not the case. Although the enzyme was quite specific for Asn at the P1 position, the enzyme tolerated considerable variability among amino acids after the conserved cleavage site. The high degree of sequence conservation, especially on the C-terminal side of the cleavage site, probably reflects structural conservation around the Cys residue at position P7'. This residue forms part of an interchain disulfide bond that links the acidic and basic chains after post-translational modification of the 11S subunits (Staswick et al. 1984 ), a linkage that is required for successful assembly of processed proglobulin trimers into 11S hexamers in vitro (Jung et al. 1997 ). The lack of a restrictive specificity by the endopeptidase toward amino acid residues on the C-terminal side of the cleavage site is consistent with the finding of a limited number of 11S subunits that contain amino acids at the P1' position other than Gly (Nielsen et al. 1997 ). These include legumins from pea (March et al. 1988 ) and several gymnosperm species (Leal and Misra 1993 ; Arahira and Fukazawa 1994 ).

Although the cocleavage assay revealed differences in the kinetics of cleavage by the endopeptidase, the procedures used in this study do not permit adequate evaluation of the effects elicited by changes in the amino acids around the cleavage site. More detailed studies with assays other than the end-point procedures that we used are necessary. Nonetheless, the evidence gathered predicts that changes in the reactions catalyzed by the endopeptidase might occur which are elicited by structural changes around the cleavage site. For example, the octapeptide that contained LeBI₃₅₆G₃₅₇ was cleaved by the endopeptidase but was not cleaved when this mutation was part of an intact subunit (Figure 4C). Interestingly, Muramatsu and Fukazawa 1993 reported results with the same kind of mutation in proglycinin G1 and found that neither a corresponding octapeptide nor the mutant subunit was cleaved by a protease from mature soybeans. The basis for the differences between our results and those of Muramatsu and Fukazawa 1993 is not obvious.

The time course of the reaction between the asparaginyl endopeptidase and both the prolegumin and proglycinin subunits provided important clues about the fidelity of the folding process that these subunits undergo after their synthesis in vitro. Some of the subunits produced in vitro are apparently incorrectly folded because they are rapidly degraded early during the course of the reaction. Later acidic and basic chains accumulate as stable products as the correctly folded subunits in trimers are processed. Experiments reported earlier by Dickinson et al. 1989 indicated that proglycinin monomers but not trimers were incorporated into hexamers upon dissociation and reassembly of native glycinin isolated from seed. Therefore, it is not surprising that misfolded subunits can be accommodated within proglobulin trimers that are assembled in vitro. What is of interest is the rapid rate at which the misfolded subunits are degraded and the slower rate at which the correctly folded subunits in trimers are processed.

Also of interest is the observation that trimers of the Met-enriched mutant 11S subunits are unstable in the presence of the asparaginyl endopeptidase, both in vitro and in vivo. Assuming extensive homology between 7S and 11S molecules (Argos et al. 1985 ; Lawrence et al. 1994 ), all of the modifications responsible for the glycinin mutants tested in this study were directed toward the hypervariable region that joins the two central ß-barrels in the molecule. Each mutant that was tested contained a short insertion in the G4 subunit (Lago et al. 1991 ). Substantial natural variation in length occurs in this region among the five glycinin subunits that have been characterized (Nielsen et al. 1989 ). Despite the natural size variation of the hypervariable region, mutations introduced into this region apparently cause conformational changes that expose asparaginyl residues sensitive to the endopeptidase. Even more restricted mutations, such as in LeBI₂₈₂G₂₈₃ and G4Q₃₅₇, can also apparently change the subunit conformation such that they are rapidly degraded. Together, our results speak to the environment in the vacuoles into which storage proteins are accumulated. The storage proteins appear to have evolved to be stable in this hostile environment, and changes that perturb their structure and expose normally buried sites that then become sensitive to proteolysis will result in digestion of the proteins. This notion has particular relevance for attempts to engineer seed proteins with improved quality characteristics. Attention must be given to whether proteins introduced into the vacuoles will be stable to the collection of enzymes in that organelle. In this regard, it is important to point out that the vacuole can be expected to contain other proteases besides the asparaginyl endopeptidase. A recent survey of seed proteins (Nielsen et al. 1997 ) indicated that proteins accumulated in seed are post-translationally modified by cleavage directly following a number of amino acids besides asparagine.

Our observations provide insight into why some proteins have been introduced successfully into seed of recombinant plants, whereas others have not. A number of modifications (Lago et al. 1991 ; Jung et al. 1993 ; Saalbach et al. 1995a ), including those reported here, made use of storage proteins that normally are accumulated in seed in large amounts. In most cases, these have been degraded. However, other proteins, such as the Brazil nut albumin (Altenbach et al. 1989 , Altenbach et al. 1992 ; Saalbach et al. 1995b ) and sunflower sulfur-rich albumin (Molvig et al. 1997 ), are accumulated to respectable levels in recombinant seed. These latter proteins are found in seed of the plants from which they are derived and are not modified structurally. It is likely that these proteins were stable in proteolysis because they were in a vacuolar environment similar to that in the plants from which they were derived. Therefore, the stability of proteins in the environment into which they will be inserted is a critical factor to consider during attempts to increase nutritional quality by either the structural modification of endogenous seed proteins or the transfer of existing seed protein genes from one plant to another. Perhaps the engineering of Met-rich 7S and 11S globulins, respectively, by Saalbach et al. 1995a and Takaiwa et al. 1995 was successful because these mutants remained stable in the vacuolar proteases.

Another potential function of the asparaginyl endopeptidase is indicated by the instability of the mutant glycinin subunits both in vitro and in vivo. That they are degraded suggests that the enzyme plays a role in editing the contents of the vacuole and serves to eliminate those proteins that are transported to the vacuole but were either misdirected or misfolded. Such a mechanism seemingly would be of benefit to the cell in protecting the function of the organelle from perturbations by foreign proteins.

Both Barton et al. 1982 and Chrispeels et al. 1982 speculated that post-translational modification of 11S proglobulins might be important for assembly of the hexamers typically isolated from legume seed. Dickinson et al. 1989 first described evidence supporting these speculations. They showed that glycinin hexamers isolated from seed could be reversibly dissociated by manipulation of ionic strength and that proglycinin trimers were unable to be incorporated into reassembled hexamers. Treating the trimer subunits with papain before reassembly could relieve this apparent block toward hexamer assembly. In contrast to treatment with papain, which causes multiple cleavages in each subunit (Dickinson et al. 1989 ), treatment of LeB or G4 subunits with the asparaginyl endopeptidase results only in those cleavages that occur in vivo. When treated in this manner, trimers composed of mature subunits that contain acidic and basic chains reassemble into hexamers in vitro. In addition, when the accumulation of LeB P1N₂₈₁ subunits in transgenic tobacco is monitored, it is possible to demonstrate that cleavage is required for assembly into hexamers in vivo as well as in vitro.

Thus, our results clearly establish the importance of post-translational modification in the assembly pathway for 11S proteins. The proteolytic modifications probably cause structural changes in the proteins that promote their assembly into hexamers. Hexamers formed from cleaved proglobulin trimers resist further cleavage by the processing enzyme. The cleavage is likely a prelude to further packaging of the proteins in the vacuole. It would be of considerable interest to know how hexamers are assembled into more complex structures within the storage vacuoles and how the deposition of 11S proteins is coordinated with that of the other storage proteins that accumulate in storage vacuoles.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Purification of Soybean Asparaginyl Endopeptidase
Purification was performed as described by Scott et al. 1992 , with the following modifications. Crude extracts were prepared by grinding 20 g of midmaturation seed in 15 mL of 0.7 mM citrate-phosphate, pH 7.0, that contained 0.4 M NaCl and 0.02% phenylmethylsulfonyl fluoride (PMSF) (extraction buffer). After centrifugation at 15,000g and aspiration of supernatant, the pellet was re-extracted with the same volume of extraction buffer and sedimented. Supernatants were combined, filtered through Miracloth (Calbiochem, La Jolla, CA), and centrifuged at 55,000 rpm in a 70Ti rotor (Beckman Instruments, Fullerton, CA) for 1 hr. The cleared crude extract was then loaded onto a 500-mL Sephacryl S300 column (Pharmacia, Uppsala, Sweden). From this point, the original protocol was followed without further deviation. Purified and concentrated protease preparation was stored at either -20 or -80°C in 0.01 M citrate-phosphate, pH 5.0, that contained 50% glycerol (MB grade; Boehringer Mannheim, Indianapolis, IN), 0.02% PMSF, 0.001% pepstatin, 0.01% leupeptin, 5 mM EDTA, and 2 mM DT T. The final preparation retained >50% of the initial protease activity for at least 6 months.

Plasmid Construction and in Vitro Mutagenesis
The plasmid pLeMS contains the field bean prolegumin B coding region (Scott et al. 1992 ). Construction of its cleavage site deletion mutant derivative pLeBN₂₈₁ was described by Saalbach et al. 1991 . Plasmids coding for the various cleavage site mutant proglobulins shown in Figure 4C and Figure 4D were produced by the method of Jung et al. 1992 . Primers used for the mutagenesis had lengths of 21 to 30 nucleotides with theoretical melting temperatures >70°C. To facilitate screening of the mutants, restriction sites were either introduced into or removed from the primers. To ensure that the mutations were correct, all mutants were subjected to DNA sequencing (Sanger et al. 1977 ) for >200 flanking base pairs around the site of mutagenesis.

Synthesis and Analysis of Synthetic Octapeptides
The octapeptides containing cleavage site amino acid residues and their variants (Figure 4B) were produced with a synthesizer (model 430A; Applied Biosystems, Foster City, CA). Digestion of the octapeptides by asparaginyl endopeptidase was performed as described by Scott et al. 1992 .

Synthesis of Proglycinin, Prolegumin, and Their Mutants in Vitro
Proglobulins were synthesized in vitro by transcription and translation, as described by Dickinson et al. 1987 , except that T7 RNA polymerase was used to transcribe DNA from pLeMS and its derivatives. Translation in a rabbit reticulocyte lysate (Promega, Madison, WI) was performed in the presence of 8 µM ³H-leucine (150 Ci/mmol; Amersham, Arlington Heights, IL), and a 10-fold concentrate was obtained from the originally supplied ³H-leucine with a SpeedVac (Savant, Holbrooke, NY) immediately before use. For the cocleavage experiments, propeptides were labeled additionally with ¹⁴C-leucine (30 mCi/mmol; Amersham), ³H-isoleucine (100 Ci/mmol; Amersham), ¹⁴C-glutamate (250 mCi/mmol; Amersham), or ³⁵S-methionine (800 Ci/mmol). To produce sufficient quantities of labeled trimers, translation reaction mixtures were usually scaled up to 20-fold. Synthesized proteins were permitted to self-assemble, and the products were analyzed by sucrose density gradient centrifugation and by trichloroacetic acid precipitation (Dickinson et al. 1987 ). The 9S trimer fractions determined from scintillation counting were pooled and concentrated to ~50 µL in a microconcentrator (model Centricon-30; Amicon, Beverly, MA) pretreated with acetylated BSA (Promega). During concentration, the buffer was changed to 0.035 M phosphate, pH 7.0, that contained 0.4 M NaCl. The final specific radioactivity in each purified and concentrated trimer preparation, as determined by direct scintillation counting, was as follows: ³H-leucine, 2000 to 100,000 cpm/µL; ¹⁴C-leucine, 1500 to 3000 cpm/µL; ³H-isoleucine, 110,000 cpm/µL; ¹⁴C-glutamate, 1000 cpm/µL; and ³⁵S-methionine, 22,000 cpm/µL. In some cases, monomer fractions were also pooled and concentrated similarly.

Digestion of Proglobulin Trimers by Asparaginyl Endopeptidase
Each reaction of total volume of 6 µL contained 1 µL of purified trimers labeled with ³H-leucine (2000 to 20,000 cpm), 1 µL of purified protease, 3 µL of 0.01 M citrate-phosphate, pH 5.0, and 1 µL of 0.035 M phosphate, pH 7.0, that contained 0.4 M NaCl. The final pH of the reaction mixture was 6.0. The mixture was incubated at 30°C for 90 min. As controls, unmodified proglobulin trimers and heat-inactivated asparaginyl endopeptidase that had been incubated at 65°C for 10 min were used. Products from the digestion were analyzed as described by Scott et al. 1992 .

For digestion of partially assembled proglobulins, 5-µL aliquots of in vitro–synthesized prolegumins were removed at various time points of incubation during self-assembly, mixed with 2 µL of asparaginyl endopeptidase, and incubated at 30°C for 90 min. The reactions were stopped by adding 7 µL of 2 x SDS sample buffer (Laemmli 1970 ) and immediately heated at 95°C for 3 min before being analyzed in a gel.

Cocleavage Assay
Purified ³H-leucine–labeled G4 trimers (22,500 cpm by tritium channel and 1350 cpm by carbon-14 channel) and purified ¹⁴C-leucine–labeled G4Q₅₅ mutant trimers (22,050 cpm by tritium channel and 15,000 cpm by carbon-14 channel) were mixed in 0.035 M phosphate, pH 7.0, that contained 0.4 M NaCl to a volume of 12 µL. To this, 12 µL of purified asparaginyl endopeptidase, 16 µL of 0.01 M citrate-phosphate, pH 5.0, and 5 µL of water were added, and the final mixture was incubated at 30°C. After 10, 50, and 90 min of incubation, 15-µL aliquots were removed, mixed with 5 µL of 4 x SDS sample buffer, and heated at 95°C for 3 min. The cleavage products and standards (unmodified protein of 7500 cpm, mutant protein of 7350 cpm, and their 1:1 mixture of 14,835 cpm as measured by hydrogen-3 channel, respectively) were separated by SDS-PAGE. After electrophoresis, the gel was treated with EN³HANCE (New England Nuclear, Wilmington, DE), dried, and exposed to film. Peptide bands detected on the fluorograph were excised and submerged in Ecolite scintillation cocktail (ICN, Costa Mesa, CA) for 24 hr. Radioactivity emitted from each peptide band was determined by scintillation counting using tritium and carbon-14 channels, respectively. The amount of uncleaved and cleaved proglobulins at each time point (represented in percentages) was calculated with the following formulas:

Reassembly of Processed Proglobulins
Purified ³H-leucine–labeled proglobulin trimers (~100,000 cpm) in 5 µL of 0.035 M phosphate buffer, pH 7.0, containing 0.4 M NaCl were mixed with 2.5 µL of 0.01 M citrate-phosphate, pH 5.0, 2.5 µL of 0.02 M citrate, and 3 µL of purified asparaginyl endopeptidase. The mixture was incubated at 30°C for 3 hr. After digestion, 15 µL of water and 5 µL of 0.175 M phosphate, pH 7.0, containing 0.4 M NaCl, together with 5 µL of 5 mg/mL dissociated glycinin (Dickinson et al. 1989 ) were added. The mixture was vortexed, and an additional 15 µL of water, 5 µL of dissociated glycinin, and 5 µL of 0.175 M phosphate, pH 7.5, containing 0.4 M NaCl, were quickly added. The final mixture was incubated at 25°C for 15 hr. After reassembly, products were separated by sucrose density gradient centrifugation (Dickinson et al. 1989 ). One-third of each gradient fraction was assayed by direct scintillation counting. The 3S and 9S fractions determined by the radioactivity counting were pooled, supplemented with 10 µg of cytochrome C, and precipitated with 10% (w/v) trichloroacetic acid. The precipitates were analyzed by SDS-PAGE (Scott et al. 1992 ).

Plant Transformation
Agrobacterium tumefaciens–mediated transformation of tobacco with DNAs encoding legumin B and its cleavage site mutant (LeBN₂₈₁) was performed as described by Saalbach et al. 1991 . Transformation and expression of the genes were verified by DNA and RNA gel blotting (Saalbach et al. 1991 ). Genes in transgenic tobacco that produced soybean glycinin were derived from plasmids pSP65/248, pSP65-RM5, and pSP65-RM6 (Dickinson et al. 1990 ). Plasmid psP65/248 contains an intact G4 proglycinin coding region. Plasmids psP65-RM5 and pSP65-RM6 are methionine-enriched mutant derivatives of pSP65/248 (Dickinson et al. 1987 ). Each of the coding regions was ligated to a glycinin-4 promoter and used for Agrobacterium-mediated transformation, as described elsewhere (Lago et al. 1991 ).

Analysis of Extracts from Tobacco Seed
Extraction of globulin preparations from transgenic tobacco seed was performed as described by Jung et al. 1993 . The extracts were concentrated, and the proteins were separated by SDS-PAGE (Laemmli 1970 ). Blotting of the protein gel and hybridization with legumin B–specific antibodies were performed according to Towbin et al. 1979 and Saalbach et al. 1991 , respectively. Centrifugation of the globulin extracts in a 6 to 22% linear sucrose density gradient and analysis of fractions by SDS-PAGE and protein gel blotting were conducted as described by Jung et al. 1993 .

	FOOTNOTES

¹ Current address: Trait and Technology Development, Pioneer Hi- Bred International Inc., 7300 N.W. 62nd Ave., Johnston, IA 50131-1004.
² Current address: USDA-ARS, Department of Agronomy, Iowa State University, Ames, IA 50011.
³ Current address: Department of Plant Pathology and Microbiology, Texas A & M University, College Station, TX 77843.
⁴ Current address: Department of Biochemistry, School of Medicine, Yeshiva University, New York, NY 10461.

	ACKNOWLEDGMENTS

We thank Drs. Larry Dunkle and Mark Hermodson for critically reading the manuscript and Jean Galbraith for help with editing. This research was supported in part by the U.S. Department of Agriculture–National Research Initiative Competitive Grant No. 91-37307-6517 and the American Soybean Association Grant No. SPR-2305 to N.C.N.

Received August 1, 1997; accepted December 23, 1997.

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