* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Proteases and proteolytic cleavage of storage
Survey
Document related concepts
Phosphorylation wikipedia , lookup
Signal transduction wikipedia , lookup
Magnesium transporter wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Protein domain wikipedia , lookup
Protein folding wikipedia , lookup
Protein phosphorylation wikipedia , lookup
List of types of proteins wikipedia , lookup
Protein (nutrient) wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Protein structure prediction wikipedia , lookup
Protein moonlighting wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Western blot wikipedia , lookup
Transcript
Journal of Experimental Botany, Vol. 47, No. 298, pp. 605-622, May 1996 Journal of Experimental Botany REVIEW ARTICLE Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds K. Miintz1 Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany Received 25 July, 1995; Accepted 16 January 1996 Abstract Proteolytic cleavage plays an important role in storage protein deposition and reactivation in seeds. Precursor polypeptides are processed by limited proteolysis to mature subunits of reserve proteins in storage tissue cells of developing seeds. Steps of proteolytic processing are closely related to steps in intracellular protein transfer through the endomembrane system and to the deposition in the storage vacuole. In germinating seeds special endopeptidases trigger storage protein breakdown by limited proteolysis. The induced conformation changes of storage proteins open them to attack by additional endo- and exopeptidases which degrade the protein reserves completely. Proteases that catalyse limited cleavage or complete degradation are synthesized as precursors which also undergo stepwise limited proteolysis when they are formed in cotyledons of developing or germinating seeds. In general, this processing transforms enzymatically inactive proenzymes into active proteases. Different compartments participate in the processing steps. Many of the proteases are encoded by small multigene families. Different members of the corresponding protease families seem to act during seed development and germination. Proteolytic processes that contribute to the molecular maturation and to the reactivation of storage proteins in dicotyledonous seeds seem to be controlled by (1) differential expression of members of the proteaseencoding gene families; (2) stepwise processing and activation of protease precursor polypeptides; (3) transient differential compartmentation of precursors and mature polypeptides of proteases and storage proteins, respectively; and (4) interacting changes in storage protein structure and protease action. The present knowledge on these processes is reviewed. ' Fax: +49 03 94 82 5-366. O Oxford University Press 1996 Key words: Dicotyledons, seeds, storage proteolytic cleavage, proteases. proteins, Introduction Developmental^ regulated changes in cellular protein patterns result from the degradation of proteins which are no longer needed, as well as from the biosynthesis of new proteins. The latter depends on the supply of amino acids which, at least in part, are recycled from protein breakdown. Similarly, cellular protein patterns change in response to environmental influences, like temperature stress (heat shock proteins), water supply (desiccation proteins), or pathogen attack (pathogenesis related (PR) proteins). Developmentally and environmentally induced changes in the cellular proteins take place at qualitative (pattern changes) as well as quantitative levels, for example, increase or decrease in enzyme activity by changing the amount of the corresponding protein. Protein biosynthesis and degradation represent important factors in the regulation of nitrogen sink/source relationships which are controlled by developmental as well as environmental factors, such as the storage and reactivation of vegetative storage proteins in soybean (Staswick, 1990, 1994) or storage proteins in seeds (Mflntz, 1994). Misfolded and damaged proteins have to be continuously eliminated by protein degradation and to be replaced by newly formed proteins. In addition, limited proteolysis contributes to precursor protein maturation and modification which often are related to intracellular protein sorting and targeting processes, like the detachment of signal peptides from precursor proteins that are synthesized at membrane-bound polysomes and co-translationally sequestered into the lumen of the endoplasmic reticulum (ER). Limited proteolysis and complete polypeptide degradation are closely interacting processes. 606 Muntz Detached protein fragments like signal and additional targeting peptides of secretory and vacuolar proteins, of nuclear-encoded plastid and mitochondria! proteins or the propeptides which result from the subsequent processing of various precursor polypeptides, for example, from the activation of zymogens, undergo rapid breakdown. The major part of these protein precursors, however, remains functionally intact and might be further transported into other cellular compartments. Proteases, subclassified into endo- and exopeptidases, catalyse limited and complete proteolysis (for reviews see Barrett, 1994; Rawlings and Barrett, 1993). Different sets of proteases and protein degradation systems have been associated with different cellular compartments (Vierstra, 1993) like the ubiqutin-dependent protein breakdown system and the proteasomes in the cytosol or the plastid protein degradation system. A system should exist to degrade glyoxysomal proteins when these microbodies are transformed into peroxisomes in lipid storing cotyledons of germinating seeds. Vacuoles in storage tissues of seeds, tubers or stems of trees harbour compartmentspecific sets of proteolytic enzymes that play a critical role in processing and breakdown of storage protein depositions (Mtlntz et al. 1985; Shutov and Vaintraub, 1987; Wilson, 1986). Seed storage proteins in the restricted sense are globulins which are characteristic of dicotyledonous seeds and a few cereals, like oats and rice, and the prolamins and glutelins from cereal grains acccording to the classification of Osborne (1924). They are only formed in specific storage tissues (e.g. endosperm or cotyledon mesophyll) during seed development. At the time of storage protein accumulation the respective organs and storage tissues represent nitrogen and carbon sinks. They become nitrogen and carbon sources when the protein reserves are reactivated. Within the frame of changing sink/source relations storage protein metabolism represents a special example for the developmentally and environmentally controlled formation and degradation of proteins. In the storage tissue cells the storage vacuole harbours the majority of participating compartment-specific proteolytic enzymes. Most storage proteins are multimeric. The subunits are polymorphic and encoded by multigene families. The primary translation products of storage protein mRNA represent precursors of the subunits formed at membranebound polysomes (rough endoplasmic reticulum, rER). Most of the precursor polypeptides (e.g. storage globulins) are transported into the storage vacuole. On their way they pass the Golgi apparatus, where at the trans-Golgi they are entrapped into transfer vesicles which are then targeted to the vacuole (for reviews see Chrispeels, 1991; Chrispeels and Raikhel, 1992; Vitale et al, 1993). As protein deposition proceeds the storage vacuole differentiates into protein bodies. In the endosperm of developing maize kernels the protein bodies are directly formed from the prolamin (zein)-accumulating ER (Larkins and Hurkman, 1978). It has also been postulated that the prolamin (gliadin) in wheat is directly transferred to storage vacuoles without passing the Golgi apparatus (Galili et al, 1995). Several of the globulin polypeptides are modified by glycosylation in the ER and Golgi apparatus and/or undergo additional processing by limited proteolysis in the vacuole. This latter step of molecular maturation then transforms the molecular species which fits to intracellular transport into a species which can be deposited in the vacuole. Clearly, the processing of precursors of storage globulin subunits by limited proteolysis is closely related to changing structure function relations which evolutionarily have been adapted on the one side to the constraints acting during intracellular protein transfer and targeting as well as on the other side to the constraints governing deposition and accumulation inside the storage vacuoles. This includes controlled limited proteolysis as well as the protection against premature storage protein degradation which might be mediated by special structural characters of the storage proteins, by maintaining proteases in an inactive state and/or by appropriate compartmentation. A third group of constraints which have most certainly played a role in the evolution of these proteins, as well as the proteases that degrade them, are those imposed by storage protein reactivation. The storage proteins must be made susceptible to proteolytic attack by appropriate structural changes, protease activation and changes in the localization of various components among various compartmentations. Limited proteolysis is essential for the initiation of storage protein breakdown. The triggering endopeptidases may be present, in an inactive state with their substrates inside the protein bodies or they may be newly synthesized during seed germination. The structural changes induced by the initial cleavages of the storage proteins result in conformation changes that open them to further degradation. In storage tissue cells this is accompanied by the reformation of the vacuole from protein bodies. The processes of storage protein deposition and reactivation have been most extensively investigated using globulins of dicotyledonous plants. Close analogues to the globulins exist in the major storage proteins of oats and rice, and in minor storage proteins of other monocotyledonous seeds. A knowledge of the structure of the storage globulins is essential for understanding their interactions with the proteases that degrade them. Globulin structure According to their sedimentation characteristics in sucrose gradients dicotyledonous storage proteins are classified into 1 IS, 7S and 2S proteins. Whereas 1 IS and 7S proteins Proteolysis of seed storage proteins belong to the globulins, except rice where the 1 IS protein was classified as a glutelin, the 2S class comprises globulins as well as albumins. The 11S and 7S globulins are named legumin-like or legumins and vicilin-like or vicilins, respectively, in accordance to the predominating storage globulins in pea (Pisum sativum L.) and faba beans (Vicia faba L.). Trivial names, which frequently have been derived from the botanical name of the corresponding plant, are in use for many of these storage proteins. Legumins (11S globulins) Legumin holoproteins, which have a molecular weight of 300—400 kDa, are composed of six nearly identical subunits with molecular weights of 50-60 kDa. Each subunit is composed of two differently sized polypeptide chains (Fig. 1). The larger more hydrophilic one (mol. wt. 30-40 kDa) has a weak acidic pi and is named a-chain, whereas the smaller more hydrophobic one (mol. wt. 20 kDa) has a strongly basic pi and is named /-chain. Both chains are linked by a disulphide bridge between cysteine residues at highly conserved positions in the a(amino acid residue 87) and /-chain (amino acid residue 7). The amino acid sequences of /-chains are more homogeneous than those of the a-chains which vary considerably in length because of different numbers of repeats in its C-terminal part (Heim et al, 1989, 1994; Horstmann et al, 1993). In addition, much larger achains are present in a minor group of large legumin subunits which, for example, have been found in seeds of Vicia faba L. The sequence of one of these large subunits (mol. wt. 64 502 kDa) supports the hypothesis that the larger subunits have evolved through amplification and mutation of repeats in the C-terminal domain of the achain (Heim et ai, 1994). There is no indication that the legumin holoprotein molecules represent homo-oligomers. In the quaternary structure of the holoprotein, two trimers are layered upon and twisted against each other at an angle of 60°. The hydrophobic /-chains are mostly buried inside the holoprotein. The more hydrophilic a-chain is mainly located at the surface and forms loops which extend out of the protein. The major loop is formed by the variable and hydrophilic C-terminal region of the a-chain. Legumin genes and the corresponding mRNAs encode subunits. Therefore, the primary translation product corresponds to one subunit. It has a transient N-terminal signal peptide. a- and /-chains are linked by a peptide bond between the C-terminal amino acid residue of the a-chain, which always is Asn, and the N-terminal residue of the/-chain, which is almost always Gly. The disulphide bridge between both the a- and /-chain regions of the legumin precursor is already formed in the lumen of the ER into which the growing pre-prolegumin is inserted during biosynthesis. The signal peptide is 607 co-translationally detached and thereby prolegumin is generated. Prolegumin assembles into trimers. These represent the transport-competent molecular form of legumin precursors (Fig. 3). The peptide linkage between a- and /-chains is only proteolytically cleaved after the prolegumin trimers have reached the storage vacuole. Since both chains have already previously been bridged by the disulphide linkage, a- and /-chains which are encoded by one gene remain paired in the subunit. Prolegumins do not seem to be able to assemble directly into hexamers. Cleavage of the -Asn-Gly-linkage forms the prerequisite for the transformation of two trimers into one hexamer. Disassembly of the subunits does not seem to occur during the trimer to hexamer transformation which results from a very specific single proteolytic cleavage. Vicilins (7S globulins) and their structural similarities to legumins Vicilin holoproteins which have molecular weights between 150 and 240 kDa are trimers composed of two types of subunits, large convicilin-like ones with mol. wt. 70-80 kDa and small subunits of about 50 kDa (Fig. 1). Homo-oligomeric as well as hetero-oligomeric trimers are known (Thanh and Shibasaki, 1977), the latter composed of different ratios of large and small subunits (Mtintz et al, 1986). Amino acid sequences of the large and small subunits are very homologous. Close to their N-terminus the large subunits contain an approximately 20 kDa large, strongly hydrophilic insertion comprising repetitive elements. The major C-terminal 50 kDa fragment exhibits high sequence similarity to the small subunits (Doyle et al, 1986). The small subunits are polymorphic and differ with respect to two characters: the degree of glycosylation and the presence of sites for limited proteolysis by trypsin-like enzymes. Non-glycosylated subunits free of cleavage sites have also been described, for example, the major 50 kDa subunit from Vicia faba vicilin (Bassuner et al., 1987; Weschke et al., 1988) and the 70 kDa convicilin subunit from peas, Pisum sativum L. (Newbigin et al., 1990). Phaseolin, the best-known 7S globulin from garden bean (Phaseolus vulgaris L.) is composed of non-cleavable 50 kDa subunits with different degrees of glycosylation (Slightom et al, 1985). There is strong evidence that vicilin and legumin have a common evolutionary origin (Shutov et al, 1995) and that their subunits have principal similarity in the 3-dimensional structure (Lawrence et al, 1994; Shutov et al, 1995) which corresponds to the structure of phaseolin from the garden bean (Lawrence et al, 1990, 1994) and canavalin from jackbean, Canavalia ensiformis (L.) DC. (Ng et al, 1993). Like legumin subunits the vicilin subunits are encoded by multigene families. Primary vicilin translation products bear transient N-terminal signal sequences which are 608 Miintz no. processing scheme subunit name plant species ( processing enzyme ) 1 N a p C I , subunits of tegunwv the 12Sgfobulra several lecteis Pimm satnnjm, victa laba. Glyctne max, Lupnus spec , Avena stbva. Oryza sattva P&utn sMtrwm, Rianus communs (signal pepWase. legumam) 2a zeffi, hocdein victtn Pfl-congiyami Zoa mays. Homeum yulpanj Gtyctne max (signal pepddase ) pnateoin vtafin 2b Phasa&us wtgam, Paum saUvum, Victa laDa (signal peptxjase) Pisum sxtrvum, Viaa fatoa 2c {signal popbdase, unknown enzymes ) a ++p p T iL 3a N c 1 convicHln Paum satiwm Glyanemax i 1 a- fl-congtyanm o-p-cooglydtwi i 1 a-gtobulln 1 (signal pepooase. protsaseCI) m 3b 1 1 1 N 1 N | m c 1 (a c 1 c 1 naptn, 2Sa«xjniin Gossyptum twsutum Brasses napus Bertnofctto excelsa N c 1 NC ^1 \ pepteJaw. legumatn, unknown enzymes?) 1 N c. 1 I —i /A\ C N "0 NC / \ r \ N C concanavalmA Canavaia enstfotmrs (signal peptidase. loguman ) Fig. I. Processing of different storage proteins by limited proteolysis during seed maturation. N and C indicate the N- and C-terminus of the polypeptides: <j>. glycosylation site: the transient N-terminal signal peptide is similarly hatched in all cases, different hatchings were used for propeptides. whereas the mature chains of the subunits remained white. I, 12S globulin processing; 2a-2c, different forms of the processing of 50 IcDa subunits of 7S globulins, 3a and 3b, different forms of the processing of 70 kDA subunits of 7S globulins, 4, 2S globulin processing; 5 concanavalin A processing. Proteolysis of seed storage proteins co-translationally detached when the growing polypeptide is sequestered from the membrane-bound polysomes into the ER-lumen. No disulphide bridges are known to link 7S globulin subunits. Core glycosylation of the respective vicilin subunits takes place in the ER where trimers are also assembled. Glycosyl side-chains are trimmed and modified in the Golgi apparatus. Like prolegumin, vicilin trimers are sorted into transfer vesicles at the trans-Golgi network and transported into the storage vacuoles. 2S storage proteins This is a heterogeneous group of proteins where polypeptides predominate which are structurally related to the napin-like 2S proteins from Cruciferae (Shewry and Tatham, 1990). 2S storage proteins are polymorphic and encoded by multigene families. The proteins are monomeric and consist of disulphide-linked large and small polypeptide chains (Fig. 1) which are derived from a common precursor which bears a transient N-terminal signal peptide. After its detachment the primary translation product undergoes an even more complicated proteolytic processing than prolegumin. In addition to the two fragments which correspond to the mature large and small polypetide chains, the precursor comprises a propeptide located between the signal peptide and the N-terminus of the small polypeptide chain. The large chain forms the C-terminal fragment in the precursor. The propeptide cleavage site as well as the cleavage site between the two polypeptide chains are flanked by Asnresidues in the P-position of many 2S protein precursors. In the ER, the regions of the propolypeptide which correspond to the small and large chains are already linked by disulphide bridge formation. After the propeptide is cleaved off, the peptide linkage between the small and large chain is cleaved. In the case of the 2S albumin of Brazil nut (Bertholletia excelsa H.B.K.) this step involves the elimination of a short linker peptide between the small and large chains. Finally, the large chain of the Brazil nut 2S albumin is trimmed by the detachment of short oligopeptide fragments at the C-terminus. 609 peptide fragment are flanked by Asn-residues in the P-position. Processing of storage globulin precursors by limited proteolysis in developing seeds Signal peptide cleavage Precursor polypeptides of storage globulins are sequestered from their cytoplasmic site of formation into the ER-lumen, thus entering the so-called secretory pathway through the endomembrane system. This membrane transfer is mediated by the N-terminal signal sequence which is proteolytically detached after finishing its function as a targeting and membrane transfer signal. The single site cleavage is catalysed by a signal endopeptidase located in the ER membrane and acting at its inner surface (Dalbey and von Heijne, 1992). The enzyme has been extensively investigated in eukaryotic animals and yeast (Lively et al., 1994). It is a hetero-oligomeric serine endopeptidase. The cleavage site between signal peptide and N-terminus of the polypeptide chain is characterized by a specific arrangement of amino acid residues described by the -l;-3-rule according to von Heijne (1986). Since signal peptides of plant proteins are correctly detached in heterologous cell-free systems composed of the wheat germ in vitro translation system, completed with dog pancreas signal recognition particles and microsomal membranes, as well as in homologous systems with plant microsomes, a similar signal peptidase should also be present in plants (Bassiiner et al., 1984). As a result of the signal peptide cleavage a propolypeptide is formed in most cases. As far as presently known, this propolypeptide does not undergo further limited proteolysis in the ER or Golgi compartment, although there the polypeptides fold into a conformational state and oligomerize, which makes them competent for intracellular transfer through the endomembrane system and into the storage vacuole. Several storage protein polypeptides are modified by glycosylation during their passage through the ER and Golgi compartment, but this glycosylation does not seem to be a prerequisite for their intracellular transfer and targeting (Voelker et al., 1989). Concanavalin A The sequence of mature concanavalin A (con A) is not co-linear to the sequence of the precursor polypeptide derived from the corresponding cDNA (Carrington et al., 1984). After the N-terminal signal peptide is cotranslationally cleaved off in the rER, the glycosylated procon A undergoes processing. An internal glycosylated oligopeptide fragment is excised and the N- and C-terminus of the precursor are ligated in a head-to-tail position (Bowles et al, 1986; Faye and Chrispeels, 1987). In addition, small terminal oligopeptide fragments might be detached (Fig. 1). Both cleavage sites of the internal Propolypeptide processing by limited proteolysis in vacuoles The presence of N-terminally Asn-flanked processing sites is a common character of many US globulin (Lawrence et al., 1994), 2S storage protein (Hara-Nishimura et al., 19936) and even protein body membrane protein precursors (Inoue et al, 1995). The complete evolutionary conservation of this Asn at the C-terminus of the large legumin polypeptide chains indicates that it may be important for recognition by the corresponding processing enzyme. Mutation or deletion of this Asn renders 610 Milntz prolegumin uncleavable in vitro as well as in vivo. Recently, cysteine endopeptidases have been described from castor bean (Hara-Nishimura et al, 1991), soybean (Scott et al, 1992; Muramatsu and Fukazawa, 1993) and jack bean (Abe et al, 1993) that at least in vitro catalyse the Asn-specific limited cleavage of prolegumins, precursors of 2S proteins, proconcanavalin A and some other proteins. An enzyme belonging to this class of endopeptidases, but prepared from germinating vetch seeds, was able to catalyse in vitro the Asn-specific molecular maturation of prolegumin (Becker et al, 1995a), that normally occurs in developing seeds. Polypeptides which cross-react with antibodies raised against the enzyme from castor bean were also detected in other plant organs except seeds (Hiraiwa et al, 1993). Therefore, this type of enzyme may belong to a class of cysteine endopeptidases which specifically cleaves at the C-terminus of Asn residues that are exposed in a way which gives the enzyme access. Legumain in developing and mature seeds: When the cDNA corresponding to the castor bean (Ricinus communis L.) Asn-specific processing proteinase from seeds was sequenced and the complete amino acid sequence was derived (Hara-Nishimura et al, 1993a, 1995), it turned out to be a new type of cysteine endopeptidase. The sequence did not correspond with the well-known papain-like cysteine endopeptidases. The only similarity that appeared on screening the databases was to the sequence of an endopeptidase from Schistosoma mansoni, a human parasite. The Ricinus mRNA encodes a 55 kDa pre-propolypeptide that is presumably comprised of a 31 amino acid-long N-terminal signal peptide, the mature 37 kDa enzyme and an approximately 14 kDa C-terminal propeptide sequence the exact length of which is unknown since the C-terminus of the mature enzyme has yet to be determined (Fig. 2b). Antibodies have been raised against the purified native proteinase protein and used to localize the enzyme to vacuoles in cells of the maturing castor bean endosperm in which US and 2S storage proteins are also localized. This result reinforces the interpretation of previous 'grind and find' experiments in which the Asn-specific processing enzyme co-purified with the protein bodies of castor bean endosperm. The same antibody cross-reacted in immunoblots with polypeptide bands after electrophoresis of extracts from mature and early germinating seeds, too. Extracts from roots, hypocotyl and leaves of castor bean as well as from pumpkin and soybean cotyledons and hypocotyls, from mung bean roots and spinach leaves were active in the enzyme- PROTBNASE A 128 1 18 152 286 307 359 C C QGQCGSCWAFST TDLNHGVA 1 A NSWG / \ KDEL ER i ? ? VA? PROTEINASE B 24 49 77 216 265 376 ( ? ) 493 ND NYRHQ.D.CHAY EACESGS TCLGDLYS ER Fig. 2. Schematic presentation of essential characters of proteinases A and B. SP, signal peptide; PP1 and PP2, propeptides; i£, glycosylation sites; C, positions of cysteine residues; sequences of highly conserved fragments are indicated; arrows, amino acid residues of the active centre in papain which are conserved in proteinase A; ER, endopiasmic reticujum, and VA, vacuole, indicate the cellular compartments where processing (presumably?) occurs. Proteolysis of seed storage proteins specific assay which uses a synthetic decapeptide substrate containing an Asn-flanked internal cleavage site (Hiraiwa et al, 1993). The cDNA-derived amino acid sequence of the vacuolar Asn-specific processing enzyme of soybean cotyledons exhibits 77% similarity to the castor bean cysteine endopeptidase and cleaves native precursor polypeptides as well as synthetic oligopeptides like the castor bean enzyme (Shimada et al, 1994). A similar endopeptidase was purified from mature jack bean, Canavalia ensiformis L. (Abe et al., 1993). Oligonucleotides derived from partial N-terminal amino acid sequence of the enzyme have been used to obtain four different cDNA clones which suggests that isoenzymes exist which are encoded by a multigene family (Takeda et al., 1994). Since similar Asn-specific cysteine endopeptidases are present in various legumes it was named legumain (Ishii, 1994; Takeda et al., 1994) according to the current edition of Enzyme Nomenclature (EC 3.4.22.34). A method was published which now permits simple and sensitive quantitation of corresponding enzyme activities even in crude extracts (Cornel and Plaxton, 1994) using benzoyl-L-asparagine-/7-nitroanilide as the substrate with subsequent diazotization of the generated p-nitroanilide which makes the spectrophotometric measurement of this reaction product more sensitive. Polymorphism of an Asn-specific 33-33.8 kDa vacuolar 11S processing enzyme has also been reported for a cysteine endopeptidase preparation from mature soybean (Muramatsu and Fukazawa, 1993). The three isoforms were isolated which had isoelectric points of 4.94, 4.89, and 4.85, respectively. They specifically cleaved proglycinin, which was recombinantly produced in E. coli, into the mature a- and _$-chains at the C-terminus of Asn in the precursor. Glycinin is the legumin-like US storage globulin from soybean and the endopeptidase was called 'maturation enzyme'. Whereas the Asn-specific cysteine endopeptidases reported so far do not seem to be glycosylated, Scott et al. (1992) prepared a glycosylated US globulin maturation endopeptidase from soybean. The enzyme which in electrophoresis under denaturing conditions gave 3 polypeptides with relative molecular weights of 85, 63 and 23 kDa in vitro correctly processed prolegumin from Vicia faba L. as indicated by N-terminal microsequencing of the cleavage product. Octapeptide substrates were synthesized with an Asn in the P-position of the internal cleavage site, and several mutations of the Asn-residue were produced. Only octapeptides having Asn in an internal P-position were cleaved by the enzyme. This confirms its strict Asn-specificity. Several lines of evidence indicate that this new class of Asn-specific cysteine endopeptidases is the prime candidate for the propolypeptide processing proteinase that catalyses the maturation of US and 2S storage proteins in dicotyledonous seeds. (1) The enzyme has been local- 611 ized to the same compartment as the storage proteins, namely the vacuoles (developing protein bodies) of cells in storage endosperm and cotyledons of developing seeds, (2) in vitro cleavage of the corresponding storage protein precursors occurs as expected, and (3) the time-course of the appearance of the corresponding enzyme polypeptide as well as the time-course of its activity correspond with the period of biosynthesis and processing of the presumptive in vivo subtrates in developing castor bean (Cornel and Plaxton, 1994; Hiraiwa et al., 1993). Nevertheless, conclusive in vivo evidence for the suggested endopeptidase function is still lacking. More conclusive evidence could be provided if it is possible to inhibit storage protein maturation by expression of appropriate antisense-DNA constructs of the proteinase in the developing seeds of transgenic plants. Legumin processing by limited proteolysis in vitro and in vivo: An in vitro processing assay for proglycinin of soybean has been developed by combining the in vitro transcription/translation of US globulin propolypeptides followed by oligomer reconstitution (Dickinson et al, 1987, 1989) with an in vitro cleavage assay using the soybean US globulin maturation enzyme (Scott et al, 1992; Jung et al, unpublished). Prolegumin B from faba bean which was produced in vitro and could be assembled into trimers was purified by isopycnic sucrose density centrifugation and used as substrate in the cleavage assay (Nielsen et al., 1995). The results of these experiments, which at least in part are still unpublished, indicate that only correctly formed prolegumin trimers undergo the single site cleavage of the peptide bond linking the a- and jS-chain in the precursor and are thereby converted into the hexamers. Prolegumin monomers are degraded probably because they assume a conformation where the many other Asn-flanked sites in the polypeptide are not protected against attack by the maturation enzyme. This result also suggests that no disassembly of the prolegumin trimer should take place in the process of trimer to hexamer transition in vivo. Confirmation of these in vitro results have come from tobacco transformation experiments. Prolegumin without the C-terminal Asn of the a-chain could not be cleaved in transgenic tobacco seeds and formed only trimers but no hexamers (Jung et al, unpublished). Prolegumin chain fragments of various length were fused before the complete chloramphenicol acetyl transferase polypeptide (CAT). Stable a-chains were formed inside vacuoles if the constructs contained an intact a//8-chain cleavage site. The detached fragments comprising partial jS-chain sequences or the complete ^-chain fused to CAT were degraded. This suggests that these fusion polypeptides were recognized as misfolded by the vacuolar proteases. Legumin-CAT fusions with fragments shorter than the complete a-chain did not contain the a/$-cleavage site. 612 MOntz They remained outside the vacuole and were not degraded (Jung et al., 1993). This processing of prolegumin-CAT fusions could also be demonstrated in vitro. The enzyme which catalyses the correct processing as well as the enzyme responsible for the degradation of the misfolded chains should be located within the vacuole. Both activities can probably be attributed to the same Asn-specific cysteine endopeptidase. The prolegumin processing activity has been localized to vacuoles of storage tissue cells of developing pumpkin and castor bean seeds (Hara-Nishimura et al., 1987; Harley and Lord, 1985). The Asn-specific processing cysteine endopeptidase has been purified from protein bodies of castor bean endosperm (Hara-Nishimura et al, 1991). Immunohistochemical studies of this protease have localized it to the vacuoles of similar cells (HaraNishimura et al, 1993a). Prolegumin trimers, but no hexamers, are present in the ER of developing storage tissue cells in pea (Chrispeels et al., 1982), soybean (Barton et al., 1982) and pumpkin cotyledons (HaraNishimura and Nishimura, 1987). Conversely, only negligible amounts of prolegumin have been found in vacuoles of corresponding cells in which legumin hexamers predominate. All these results indicate that prolegumin trimer assembly takes place in the ER. The trimers are then transferred into the storage vacuoles where they are cleaved by the processing enzyme. This cleavage of the a//?-chain peptide linkage transforms the prolegumin trimer directly into the mature legumin hexamer which is then able to form deposits in the developing protein bodies. Formation of the processing enzyme as well as of the prolegumin are subject of strict developmental control (Hara-Nishimura and Nishimura, 1987, 1993a). Both concomitantly undergo intracellular protein transfer through similar compartments. Since no cleavage of the prolegumin seems to occur before reaching the vacuole, the protease should be inactive during its passage through the secretory pathway and becomes activated after its arrival in the storage vacuole. A finely tuned interplay of changing structure function relations between substrate and enzyme proteins, of enzyme activation and compartmentation is responsible for regulating this process. Processing of a protein body membrane protein precursor: Recently it has been shown (Inoue et al, 1995) that protein body membrane polypeptides MP27 and MP32 arise from the translation of an mRNA into a common precursor which must be post-translationally processed into the two polypeptides. The putative cleavage site in the P-position is flanked by an Asn-residue. The authors suggest that here also an Asn-specific cysteine endopeptidase may be the processing enzyme, since, as above, the precursor polypeptide enters the secretory pathway and the polypeptides are associated with the inner surface of the protein body membrane. Limited proteolysis of provicilin in vacuoles: Posttranslational cleavage of vicilin precursors has been demonstrated in developing pea (Gatehouse et al., 1981, 1982, 1983) and field bean cotyledons (Scholz et al., 1983). Some of the polymorphic 50 kDa pea vicilins contain two internal cleavage sites flanked by Arg and Lys residues, whereas others are free of such sites. In mature pea cotyledons sets of distinct polymorphic cleavage products of 33, 19, 16, and 13.5 kDa have been found which are generated by cleavage at both or only one of the two preformed sites. The 16 kDa fragment was glycosylated. In field bean 50 kDA vicilin subunits predominate that are free of such cleavage sites. The proportion of the vicilin subunits that were cleaved to sizes comparable to those found in pea was strongly dependent upon the preparation method. Since in the same legume plant cleavable as well as non-cleavable forms of 50 kDa vicilin subunits co-exist and in certain legumes, like garden beans, no cleavage of the vicilin-homologous phaseolin subunits takes place, it is difficult to assign a function to this processing step. This type of limited proteolysis does not appear to occur in the processing of convicilin-like subunits of 7S globulins. Nevertheless, these proteins are subject to specific processing. In developing cotton seeds (Gossypium hirsutum L.) the vicilin-like storage proteins, named aglobulins, are represented by two groups of polymorphic subunits, the glycosylated 52 kDa and the nonglycosylated 46.5 kDa polypeptides (Dure and Chlan, 1981). Both are generated by processing of a family of propolypeptides with molecular weights of about 67 kDa (Chlan et al., 1986, 1987). An N-terminal 20.5 kDa fragment is detached by limited proteolysis from the precursor to form the mature vicilin-like chains. The putative cleavage site is flanked by Arg-residues, so that a processing enzyme different from the prolegumin processing endopeptidase must be responsible. The intracellular site of this processing event still remains to be elucidated. Other processing enzymes? A putative aspartate endopeptidase has been partially purified from extracts of developing and mature Brassica napus seeds and used in processing experiments with in vrtrosynthesized and radioactively labelled rape napin-like 2S albumin propolypeptide from Arabidopsis thaliana as a substrate (compare Fig. 1). In addition, synthethic substrates have been used that had been designed according to the peptide which links the small and large chain in the albumin precursor and is excised during its processing (D'Hondt et al, 1993). Internal peptide bonds were cleaved in these substrates which suggests that the enzyme could also process the precursor. The propolypeptides were split into fragments that correspond to the size of the small and large chains of mature 2S albumin. Thus the authors Proteolysis of seed storage proteins claim that they have found a processing protease which at least in vitro might correctly process 2S storage albumin precursors of cruciferous seeds. No evidence was presented that the enzyme is present in a compartment where 2S proalbumin processing might take place and how the N-termini of the mature chains might be generated by this or an additional enzyme. Limited proteolysis and storage globulin degradation in germinating seeds In mature dry seeds storage proteins are present in the embryo axis as well as in the storage tissues proper, like the endosperm, for example, in castor beans, or the cotyledon mesophyll, for example, in legumes, oil seed rape, sunflower or pumpkin. However, in the embryo axis, time-course, pattern and mechanism of storage protein degradation as well as its contribution to nitrogen supply for the developing embryo and its regulatory interaction with the major protein degradation processes in the proper storage tissues has not been investigated. Research has been focused on proteolytic enzymes and storage protein degradation in the proper storage tissues. There, the beginning of measurable storage protein degradation can be detected at days 2-3 after the start of imbibition (dai) depending on the species under investigation. Further storage protein breakdown proceeds much more rapidly in the cotyledons of germinating Vigna radiata (L.) Wilczek. (Baumgartner and Chrispeels, 1979) or Phaseolns vulgaris L. (Nielsen and Liener, 1984; Boylan and Sussex, 1987) where 7S globulins strongly predominate, than in seeds of Pisum sativum L. (Basha and Beevers, 1975), Vicia faba L. (Lichtenfeld et al., 1979, 1981) or Glycine max (L.) Merr. (Wilson et al., 1986) in which nearly 50% or more of the storage protein is made of 1 IS globulin. Nevertheless, the overall pattern of storage globulin degradation is similar and, in parallel to storage globulin breakdown, protein bodies start to fuse and large vacuoles are regenerated in the storage tissue cells. Pattern of storage protein cleavage If globulins from field beans were analysed by electrophoresis under non-denaturing conditions or by immunoelectrophoresis slight mobility changes were observed for vicilin and legumin during the first days of germination. Changes in mobility in immuno-electrophoresis may be due to changes in the charge (Lichtenfeld et al., 1979). Similar observations were made with germinating vetch seeds (Shutov and Vaintraub, 1973), beans (Boylan and Sussex, 1987) and buckwheat (Dunaevski and Belozerski, 1989a). If samples from early germination stages were analysed under denaturing conditions, cleavage products were apparent. As germination progressed, the original 613 bands became weaker and distinct breakdown products appeared. Taken together these results suggest that limited proteolysis plays an important role in initiating storage globulin degradation. After limited proteolysis the holoproteins nearly retain their original electrophoretic mobility under non-denaturing conditions. The holoproteins also retain their sedimentation characters in sucrose gradient centrifugation. Both results indicate that at this stage the proteins are largely intact. Only very small fragments are lost at this stage. Consequently, the amount of liberated amino acids must be small. This is reflected in the slow initial decrease of protein nitrogen that was observed in germinating seeds. Whereas distinct large size cleavage products were found the small ones have never been electrophoretically demonstrated, indicating that the half-lives of the smaller fragments may be much smaller than those of the large fragments. The amino acids produced by the rapid breakdown of the small fragments are transferred to the growing germling where they provide the starting material for new protein biosynthesis. The breakdown of the major amount of storage globulins occurs from 4-8 dai depending on the plant species and it coincides with the major activity of proteolytic enzymes. Pattern of legumin degradation: In seeds containing both legumins and vicilins the degradation of legumin proceeds more slowly (Lichtenfeld et al., 1979, 1981; Wilson et al., 1986). The a-chain of legumin starts to be degraded first whereas the jfl-chain remains intact until 5-6 dai in cotyledons of germinating field bean and soybean when the major storage protein degradation occurs. This might be attributed to the topographic position of this chain in the holoprotein. The a-chains are thought to be exposed at the surface while the _/8-chains are located inside the holoprotein. This is supported by two observations. (1) Antibodies raised against the holoprotein predominantly react with a-chains, but only weakly or not at all with the jS-chains in immunoblots; and (2) in short-time in vitro incubation experiments endopeptidases nearly exclusively cleave a-chains, but not jff-chains. The major cleavage intermediates exhibit relative molecular weights of 24 000 to 30 000 which is larger than the molecular weight of j9-chains, thus indicating that these intermediates must have been generated from the a-chains. No detailed analysis of the cleavage products such as partial N-terminal amino acid sequencing has so far been performed. Therefore, no exact localization of the cleavage sites is known. In cleavage experiments with trypsin distinct fragments of the a-chain of glycinin, the leguminlike globulin from soybean, are generated (Shutov et al., 1993). The fragment sizes suggest that the sites that are attacked correspond to loops II and 12 in the predicted secondary structure of legumin (Shutov et al., 1995). Similar sites should represent the targets of the first, limited, endopeptidolytic attack during legumin degradation in germinating seeds. 614 MQntz Pattern of vicilin degradation: Conglycinin is the major vicilin-like 7S globulin in soybean. During germination the large a- and a'-subunits of ^-conglycinin disappear first (Bond and Bowles, 1983; Wilson et al, 1986). Whereas Bond and Bowles (1983) already found degradation of these polypeptides in mature and imbibing seeds, Wilson et al. (1986) have reported that the breakdown of these ^-conglycinin subunits starts at the earliest 1 d after imbibition. As the a- and a'-^-conglycinin polypeptides disappear, a 51 200 kDa polypeptide accumulates which reacts with jft-conglycinin-specific antibodies and is generated by the detachment of an N-terminal fragment from the large ^-conglycinin subunits (Qi et al., 1992). This limited proteolysis step resembles the processing reported for large vicilin subunits of cotton during seed maturation (Chlan et al., 1986). The intermediates of aand a'-jS-conglycinin degradation start to disappear at 6 dai when new jS-conglycinin-specific intermediates with mol. wt. 25-31 kDa emerge. The 50 kDa _/?-^-conglycinin chain does not seem to be degraded until 6 dai. Its degradation starts at the same time as that of the 51.2 kDa intermediate degradation product of the a- and a'-ficonglycinin. Therefore, it has not been possible to determine the precursors of the 25-31 kDa conglycinin cleavage products. The degradation of j0-j?-conglycinin coincides in time with the breakdown of the jfl-chains of glycinin, the legumin-like US globulin of soybean. In Vigna radiata and Phaseolus vulgaris, in which the major globulins are 7S trimers containing predominantly 50 kDa subunits, globulin degradation takes place around the 3rd to 4th dai (Baumgartner et al, 1979; Nielsen and Liener, 1984; Boylan and Sussex, 1987). Groups of degradation intermediates with Mt 20-30 kDa appear. The non-glycosylated 45.5 kDa phaseolin polypeptide seems to be degraded more easily than the glycosylated 51 and 48 kDa subunits (Nielsen and Liener, 1984). Germination proteases By applying different protease inhibitors to germinating castor beans (Alpi and Beevers, 1981) and to extracts from germinating garden beans (Nielsen and Liener, 1984) and mung beans (Chrispeels and Boulter, 1975) it was possible to determine that sulphydryl proteinases or, as they are named today, cysteine endopeptidases, are primarily responsible for storage globulin degradation in vivo. Numerous publications in which increases in amount and activity of cysteine endopeptidases are correlated with the major breakdown of storage globulins, support this conclusion (for reviews see Wilson, 1986; Shutov and Vaintraub, 1987; Vierstra, 1994). Some authors also report the involvement of serine endopeptidase (Mitsuhashi et al, 1986; Qi et al, 1992) or metalloendopeptidase (Belozerski et al, 1990; Elpidina et al, 1991) in storage globulin degradation. The pioneering work of Chrispeels's group (reviewed by Baumgartner and Chrispeels, 1979) on the proteolytic breakdown of vicilin in germinating seeds of Vigna radiata has demonstrated that cysteine endopeptidases synthesized de novo, in their case a vicilin peptidohydrolase (Baumgartner and Chrispeels, 1977), catalyse globulin degradation in the cotyledons of germinating legume seeds. Purified vicilin peptidohydrolase has a mol. wt. of 23 kDa, an IEP at pH 3.75 and exhibits maximal activity at pH5.1. The enzyme preferentially cleaves synthetic esters with asparagine and glutamine, readily degrades vicilin in vitro, and exhibits its major activity increase in germinating seeds from 2-5 dai when most of the vicilin disappears. It is synthesized at membrane-bound polysomes. The enzyme protein undergoes vesicular transport into the protein storage vacuole where it initiates the degradation of globulin (Baumgartner et al, 1978). Although a protease inhibitor with in vitro activity against the vicilin peptidohydrolase is present in the cotyledon cells of germinating Vigna radiata seeds, it is located in a different subcellular compartment so that it probably plays no role in the regulation of vicilin degradation (Baumgartner and Chrispeels, 1976; Chrispeels et al, 1976; Chrispeels and Baumgartner, 1978). Since isolated protein bodies from mature seeds showed only negligible endogenous protein degradation and storage protein breakdown was elicited by the addition of extracts from 4 dai seeds, the de novo synthesis of the vicilin peptidohydrolase is assumed to be the key event in vicilin degradation (Harris and Chrispeels, 1975; Baumgartner et al, 1978). This was confirmed by detailed studies on vicilin and legumin degradation in germinating vetch, Vicia sativa L. (for review see Shutov and Vaintraub, 1987). In this system two proteases participate in globulin degradation: a triggering cysteine endopeptidase A with an Afr 21 kDa (cDNA-derived mol. wt. 25 244 kDa), which is active against globulins prepared from mature dry seeds, and a cysteine endopeptidase, termed proteinase B, with Mr 38 kDa that exhibits strict Asn cleavage specificity. Both enzymes appear to be absent from the mature seed and to be de novo synthesized during germination. Since the substrate for proteinase B is globulins that have already been modified by the 'triggering' proteinase A, its activity is only apparent after proteinase A has become active. A serine endopeptidase appears to be required for the partial hydrolysis of the large vicilin-like subunits of soybean ficonglycinin, the a- and a'-jS-conglycinin chains (Qi et al, 1992). This has confirmed an earlier study that implicated a serine endopeptidase in the limited proteolysis of a 64 kDa subunit in cotyledons of germinating Vigna mungo seeds (Mitsuhashi et al, 1986). In both cases no corresponding enzyme activity could be measured in extracts from mature dry seeds. A different model for the initiation of globulin breakdown has been developed by Belozersky and his Proteolysis of seed storage proteins group by studying germinating buckwheat (Fagopyrum esculentum L.). They found a Zn-dependent metalloendopeptidase that forms an inactive complex with an endogenous protease inhibitor inside the protein bodies of mature seeds (Belozerski et al, 1990; Elpidina et a!, 1991). The enzyme was shown to degrade in vitro a legumin preparation from mature buckwheat. They proposed that the dissociation of the enzyme from the inhibitor which is mediated by zinc ions, triggers globulin breakdown. In addition, a cysteine endopeptidase has been isolated from germinating buckwheat which degrades legumin prepared from seedlings, but not that prepared from dry seeds (Dunaevsky and Belozersky, 19896). The authors proposed that limited proteolysis of legumin by the metallo-endopeptidase, which is already present in mature seeds, forms a prerequisite for further degradation of legumin by the sulphydryl endopeptidase and that the activity of the latter protease is feedback inhibited by degradation products. Papain-like cysteine endopeptidases: proteinase A: Papainlike cysteine endopeptidases represent the major storage protein degrading enzymes which appear during seed germination, for example, in cotyledons of Phaseolus vulgaris (Boylan and Sussex, 1987), of Vigna mungo, where it is called SH-EP (Akasofu et al, 1989, 1990) as well as of Vicia sativa (Becker et al, 19956), where the enzyme is named proteinase A. A more distantly related cysteine endopeptidase protein, termed P34, has been found in the cotyledons of developing, mature and germinating soybean, Glycine max (Kalinski et al, 1990, 1992) although evidence is still lacking that it is enzymatically active. A cysteine endopeptidase from Vicia faba also seems to belong to this papain-like class of proteinases (Yu and Greenwood, 1994). Only very late during germination it reaches maximum activity after approximately 50% of the stored proteins had already disappeared. A member of this enzyme group has even been found in pods of developing Phaseolus vulgaris fruits. It shows more than 90% sequence similarity to the Vigna mungo SH-EP (Tanaka et al., 1991, 1993). Homologous storage protein degrading endopeptidases have been found in other dicotyledonous seeds as well as cereal grains, like barley and rice, during germination. Proteinase A, SH-EP, and P34 are synthesized at the rough endoplasmic reticulum (rER) and eventually appear in the protein storage vacuole. The primary translation products have mol. wts similar to or larger than 43 kDa and undergo four to five limited proteolytic cleavage steps (Fig. 2). The first step is the detachment of the N-terminal signal peptide during translation which yields proproteinase A, a proteinase A precursor polypeptide, that is comprised of an N-terminal propeptide of approximately 120 amino acid residues, and a fragment which corresponds to the mature enzyme. The N-terminal 615 propeptide is detached by two to three consecutive cleavage steps. Whereas the proproteinase SH-EP and its first cleavage product are inactive, the product of the next cleavage exhibits very low activity. The enzyme becomes fully activated by the last proteolytic processing step(s) (Mitsuhashi and Minamikawa, 1989). The tetrapeptide KDEL which is present at the C-terminus of cDNAderived sequences of SH-EP and proteinase A precursors is known to be a ER retention signal. It is lacking in isolated-mature SH-EP (Okamoto et al., 1994) and proteinase A (Becker et al., 19956) of germinating seeds. One could speculate that this step coincides with the transfer of the proenzyme from the ER to the vacuole and/or an intermediary compartment on the way to the vacuole. The mature enzymes have different molecular weights: SH-EP, 33 kDa (electrophoretically determined); P34, 28.6 kDa; and proteinase A 25.4 kDa (both calculated from cDNA-derived sequences). Exactly where in the cell the N-terminal propeptide fragments are detached is still unknown. However, for several reasons discussed later, at least the final step should take place in the vacuole. Only the propeptide regions of the precursors of proteinase A and P34 contain glycosylation sites, which agrees well to the finding that the mature enzymes are not glycosylated. The P34 precursor has been shown to be glycosylated (Kalinski et al, 1992). SH-EP also contains potential gJycosylation sites in the mature polypeptide, but it is not known whether they actually bear carbohydrate side chains. Papain-like cysteine endopeptidases exhibit optimal activity at pH values near 5. The mature enzymes have 7 cysteine residues and contain conserved elements which are known to be part of the active centre of papain (Fig. 2a). Whereas only a single gene appears to code for the Vigna mungo SH-EP (Yamauchi et al., 1992), small gene families with at least two members have been shown to code for proteinase A (Becker et al., 19956) and P34 (Kalinski et al., 1990). Proteinase P34 not only in its primary structure is less related to SH-EP than proteinase A, but it also has no C-terminal KDEL-signal for ER-retention. In mature soybeans P34 forms dimers which are thought to be an inactive state of the enzyme (Herman et al, 1990; Kalinski et al., 1992). A Cys residue at position 10 seems to be responsible for dimerization. In germinating seeds an N-terminal fragment is detached which transforms P34 into P32. It was speculated that the dimer dissociates by this step into monomers and the enzyme is activated by monomer formation. No de novo formation has been shown during germination. Other papain-like cysteine endopeptidases, termed CPR1 and CPR2, have been found in the cotyledons of germinating Vicia sativa seeds (Becker et al., 1994). They are formed de novo during germination. The greatest sequence homology so far found for CPR2 is to a drought stress-inducible pea cysteine endopeptidase that has been 616 Milntz found in vegetative organs (Guerrero et al., 1990) and to a related enzyme from developing soybean cotyledons (Nong Van Hai et al, 1995). The vetch seed enzyme can also be induced by drought stress (Fischer, unpublished). It is still unknown whether these vetch endopeptidases participate in the storage protein degradation or not. However, they become active at the time of storage globulin degradation, and, since the cDNA-derived amino acid sequences contain N-terminal signal peptides, they are probably located in the proper subcellular compartment. Sequence comparison with N-terminal sequences of homologous enzymes suggest that both cysteine endopeptidases contain propeptides that are processed to yield the mature enzyme. Calculated molecular weights of mature CPR 1 and 2 are 26.1 and 24.9 kDa, respectively, and both have activity maxima in the acidic pH-range. The conservation of elements of the active centre as well as the positions of the Cys-residues indicate that the enzymes are related to papain (see also Fig. 2a). Proteinase B: a legumain-like cysteine endopeptidase: Proteinase B has been purified from cotyledons of germinating Vicia sativa and partially sequenced. The sequences were then used to derive oligonucleotides in order to clone and sequence the corresponding cDNA (Becker et al., 1995a). The derived amino acid sequence of the enzyme indicates that it belongs to a class of cysteine endopeptidases, termed legumain (already referred to), that have recently been found in maturing castor bean and soybean as well as in mature jackbean. The enzyme strictly cleaves at sites with Asn or Asp in P-position and processes prolegumin like the legumains from developing seeds. Its pH optimum is 5.6. As is true of legumain from maturing seeds, the enzyme is synthesized as a precursor composed of an N-terminal signal peptide (putatively 24 amino acid residues long), followed downstream by a 24 residues N-terminal propeptide, the sequence of the mature enzyme and finally a 10-12 kDa C-terminal propeptide (Fig. 2b). To generate the mature enzyme, which has a mol. wt. of 37-39 kDa, the precursor has to undergo at least three proteolytic processing steps: the cotranslational signal peptide detachment, and the cleavage of the N-terminal and of the C-terminal propeptides, which are flanked by Asn residues at the cleavage sites. There are some indications that the precursor undergoes autocatalytic cleavage and activation. Two potential glycosylation sites are present in the mature enzyme. Proteinase B is synthesized de novo during vetch seed germination. Serine endopeptidase in germinating soybean: The serine endopeptidase Cl (Qi et al., 1992, 1994) of soybean which partially cleaves a- and a'-jff-conglycinin chains as well as the homologous convicilin from pea has a pH optimum at 3.5 to 4.5 and a mol. wt. of 70 kDa. Although no corresponding activity could be measured in extracts from mature seeds, the enzyme as well as jS-conglycinin degradation could already be detected after 1 d of imbibition. Distinct cleavage products with mol. wt. 48-50 kDa are generated in a stepwise fashion. The enzyme cleaves in vitro inside specific clusters of acidic amino acid residues independent of whether these are present in large vicilin subunits or in the C-terminal region of the a-chain of legumin (Qi et al., 1994). A presumably related enzyme has been found in germinating Vigna mungo seeds (Mitsuhashi el al., 1986). Metallo-endopeptidase in germinating buckwheat: A Zncontaining enzyme with a pH optimum between 8.0 and 8.2 has been purified to homogeneity in electrophoretic analysis. Its relative mol. wt. has been determined to be 34 kDa by electrophoresis under denaturing conditions and 39 kDa by gel chromatography. The enzyme cleaves peptide bonds in oxidized insulin 5-chains which contain Leu, Tyr or Phe in the P-position (Belozerski et al., 1990) and cleaves legumin prepared from mature buckwheat as well as from soybean seeds. The metallo-endopeptidase appears to be reversibly inactivated by association with an endogenous proteinase inhibitor polypeptide. It has been localized in protein bodies (Elpidina et al., 1991). Protein processing by limited proteolysis and the control of storage protein maturation and breakdown in legume seeds: an hypothesis Endopeptidase-specific antibodies were used along with the appropriate cDNA probes to detect the presence of the enzyme and mRNA in storage tissues of developing and germinating seeds and to follow the time-course of their appearance. Proteinase A- (Kalinski et al., 1992; Becker et al., 19956) and proteinase B-specific polypeptides (Hara-Nishimura et al, 1993a; Becker et al., 1995a; Okamoto et al, 1995) have so far been detected in developing as well as in mature and germinating seeds of different legumes, castor bean and pumpkin. Polypeptides with the size of mature proteinase B were found in protein bodies prepared from vetch seeds 8 h after the start of imbibition. Precursors of proteinase A could only be detected in fractions that did not contain protein bodies and they corresponded in size to precursor polypeptide bands found in developing seeds. Three days after imbibition, when both these polypeptide bands were no longer detectable on Western blots, first newly formed proteinase B mRNA appeared, closely followed by the appearance of proteinase A mRNA. No signals for proteinase A- and B-specific mRNAs have been detected at earlier germination days. Approximately 24 h later, the proenzyme bands appeared in Western blots and these were followed by the appearance of intermediate precursors as well as those of mature proteases within the next few days. This reinforces previous results which indicated that these proteases are synthesized de novo during germination. Proteolysis of seed storage proteins The proteinase B polypeptides that have been detected in the cotyledons of mature, early imbibing and early germinating seeds must have been synthesized, processed and transported to protein bodies during seed development (Hara-Nishimura et al., 1993a). On the other hand, only precursor polypeptide bands of proteinase A have been found in extracts prepared from maturing vetch cotyledons and these were absent in protein body fractions of 8 h imbibed seeds. This indicates that processing and transport of proteinase A precursors occur either very late during seed maturation or early after imbibition. This would make sense, if as presumed by Kalinsky et al. (1992) and others (Becker et al., 19956) proteinase B participates in the molecular maturation of proteinase A precursors which have the necessary Asp residue in the P-position at the processing site in front of the N-terminus of the mature polypeptide. Thus, proteinase A, which can cleave mature globulin holoproteins, would remain outside the protein bodies until storage protein degradation is induced. Proteinase B, which can not attack mature globulins, can be safely stored along with the globulins inside the protein bodies. During protein body development its activity is necessary for the maturation of prolegumin (Mtlntz et al., 1993), 2S protein precursors 617 (Hara-Nishimura et al., 1991, 1993ft) and membrane proteins (Inoue et al., 1995) at their Asn-flanked cleavage sites. In a similar way it could contribute to the final activation of proteinase A precursors during seed imbibition when these precursors are transferred into the protein bodies. Provided proteinase A-like precursors, which have been found outside the protein bodies in developing and mature seeds, have a C-terminal KDEL as found in similar enzymes of germinating seeds, this tetrapeptide may be required to delay its transfer into the vacuole. The detachment of this ER-retention signal would permit the transfer of the precursor into protein bodies where the last N-terminal propeptide could then be cleaved by action of proteinase B. If this hypothesis is correct then KDEL detachment would play an important role in controlling breakdown of globulin. In contrast to SH-EP and proteinase A the precursor of P34 has no KDEL at its C-terminus (Kalinski et al., 1990) and, as expected, it has been localized in the protein bodies of maturing soybean (Kalinsky et al., 1992). The metalloendopepidase of mature buckwheat has also been found to be located inside the protein bodies along with the buckwheat legumin (Elpidina et al., 1991). This enzyme and P34 are supposed to be inactive inside protein bodies of mature PHOTBNBOOY PROTEIN BOOT Fig. 3. Hypothetical scheme of compartmentation, processing of a storage protein (12S globulin taken as the example) and cysteine endopeptidase precursors as well as proteinase activation to integrate the data so far obtained for cotyledon cells of developing, mature and germinating vetch (Vicia saliva L.) seeds (A), for soybean (Glycine max L. (Merr.) (B), and buckwheat (Fagopvrum esculemum L.) (C). ER, endoplasmic reticulum, LEG, legumin; KDEL, C-terminal tetrapeptide, presumably acting as ER retention signal. 618 MQntz seeds. The metallo-endopeptidase of buckwheat has been found to be inactivated by its binding to a protease inhibitor (Fig. 3c) that is also present in the protein bodies (Elpidina et ai, 1991). The P34 dimer that has been detected in protein bodies of mature soybean (Fig. 3b) is thought to represent an inactive state of the enzyme. Transformation of P34 into P32 is suggested to generate monomers which are assumed to be the active enzymes (Herman et ai, 1990; Kalinsky et ai, 1992). Other proteins, together with the storage globulins, that are located inside the protein bodies have also to be protected against an uncontrolled degradation by the proteinase B-like processing enzyme. This protection could either be due to an insensitive structure, as in the case of hexameric legumin, or by subcompartmentation of the storage vacuole. The amount of proteinase A and B protein, as visualized in Western blots, is much lower in cotyledons of developing, mature and early germinating seeds than it is later on during germling growth when most of the globulin is broken down. According to the model of Shutov and Vaintraub (1987) proteinase A begins degradation of globulins by cleaving a small number of exposed surface sites following the zipper cleavage mechanism. These cleavages trigger conformational changes that make the protein accessible for proteinase B and carboxypeptidase which are both present in protein bodies of mature seeds. The low ratio of proteases to globulin early in germination may explain why only limited globulin degradation occurs at that time. Through the combined action of proteinase A, proteinase B and carboxypeptidases, the released globulin fragments might be rapidly degraded down to small peptides and even amino acids. This would explain why it has not been possible to detect cysteine endopeptidase activity and cleavage products in extracts from early germination stages. It is still unclear how important the stored cysteine endopeptidases are for storage protein breakdown since they are eventually replaced by large quantities of de novo synthesized enzymes with similar specificities. Two findings indicate that they are active. (1) early changes in mobility of the holoproteins which have been attributed to charge shifts resulting from the detachment of fragments containing acid and/or basic amino acid residues. Clusters of such residues are located in the N-terminal regions of large vicilin subunits and in the C-terminal region of a-chains from legumin. Both regions are known to be cleaved at the initiation of globulin breakdown. However, these charge shifts have also been interpreted as an effect of protein deamidation and recently a plant protein deamidase has been characterized in germinating wheat (Vaintraub et ai, 1992). (2) Proteinase A (called vignain) and B (called legumain) activity could be detected in extracts of mature seeds of Vigna aconitifolia by Kembhavi et al. (1993). Furthermore, much higher activity was measured in extracts of seeds during the first 3 dai, if the extracts were acidified. Without acidification the activities of both proteinases increased from 0 h of imbibition (hai) and peaked 36 hai with a subsequent decrease until it reached nearly zero after 72 hai. Proteases of other classes have also been reported to be present in dry seeds, among them serine endopeptidases and metallo-endopeptidases (see review by Wilson, 1986). As mentioned above a serine endopeptidase has been shown to catalyse the initial cleavages of large vicilin subunits. In this case the de novo formation of the enzyme appears to be a prerequisite for the degradation of these polypeptides (Qi et ai, 1992). According to Belozerski et al. (1990) the buckwheat metallo-endopeptidase which is assumed to initiate legumin degradation is present in an inhibitor-inactivated state in the mature seed and it becomes activated by inhibitor dissociation (Elpidina et al., 1991). The histochemical analysis of germinating mung bean (Harris and Chrispeels, 1975) and soybean cotyledons (Diaz et al., 1993) has revealed that storage protein degradation does not start synchronously in the whole storage tissue. It is slowly initiated only in subepidermal cell layers (mung bean) or in cells adjacent to the vascular bundles (soybean). From these starting sites, which can obviously be different in different grain legumes, the increase in endopeptidase activity as well as in protein degradation proceeds wave-like inwards through the tissue. The low levels of proteinase and protein breakdown activities that have been measured in extracts from seeds of these early germination stages could result from the small percentage of cells where protein reactivation has already been started in storage tissue. Therefore, initially the small amounts of stored endopeptidases should only become active in protein degradation inside the protein bodies of limited cell layers. It can even be speculated that in these cell layers enzyme de novo synthesis has already been started. The corresponding mRNA levels remain below the limits of sensitivity of Northern blots which always have been done with extracts from a tissue where the majority of cells still were inactive in transcription of endopeptidase genes. New experimental approaches are needed to elucidate the mechanisms which initiate protein reactivation in storage tissues during imbibition and early germination of seeds. Protein storage vacuoles have been thought to be formed by transformation of the vegetative vacuole in the storage tissue cells which already exists before globulin deposition starts (Chrispeels, 1991). Recently, it has been reported that in developing pea cotyledons the protein storage vacuole is formed de novo (Hoh et ai, 1995; Robinson et ai, 1995). The vegetative vacuole degenerates. In our laboratory this was also observed in developing cotyledons of Vicia narbonensis L. (Hillmer unpublished). Protein bodies seem to be generated from the storage vacuole by two different mechanisms: Proteolysis of seed storage proteins (1) budding of the storage vacuole during early maturation stages of storage protein deposition; and (2) fragmentation during later stages. In addition, it has been suggested that protein bodies could be generated directly from the ER during late maturation (Robinson and Hinz, 1995). Thus, as suggested previously by Adler and Miintz (1993), differential protein body formation seems to occur. It remains to be investigated whether the different protein bodies are similarly equipped with proteinases or not. Since small gene families encode proteinases A and B, differential gene activation in developing and germinating seeds might lead to the formation of isozymes during the different ontogenetic periods. Thus according to our hypothesis (Fig. 3) processing as well as degradation of storage globulins in the storage tissues of developing and germinating seeds might be controlled (1) by differential expression of genes encoding pre-propolypeptides of isoforms of different cysteine endopeptidases, (2) by stepwise and differential limited proteolysis of these endopeptidase precursors which finally leads to the generation of the active mature enzyme in the vacuole; (3) by transient differential compartmentation of cysteine endopeptidase precursors and activation-catalysing enzyme(s) as well as (4) by transient differential compartmentation of degradation-triggering cysteine endopeptidase and its storage globulin substrate. It is predicted that other endopeptidases contributing to storage globulin processing or degradation might be controlled in a similar fashion. In addition, specific conformational changes induced by the action of the proteases on their substrate globulins contribute to the control of processing and degradation of storage proteins. The rate of globulin breakdown may also be regulated via feedback regulation by the level of amino acids at the site of storage protein breakdown as has been established for the buckwheat cysteine endopeptidase (Dunaevski and Belozerski, 1989b). Protein reserve activation in storage tissues seems to be under the control of the embryo axis. Cotyledons detached from the axis before imbibition exhibited no increase in endopeptidase activity and globulin breakdown. The mechanisms of signal transduction between axis and cotyledons or endosperm are still under investigation (recently reviewed by Bewley and Black, 1994). Depletion of amino acids in the growing axis, which is the major site of protein biosynthesis in the germinating seed (Dunaevsky and Belozersky, 1993) as well as phytohormonal signals transmitted from the axis to the storage tissues (Gifford el ai, 1984; Nandi et ai, 1995) have been implicated in controlling the breakdown of protein reserves. Complete breakdown of storage proteins is the result of massive de novo proteinase formation and the combined action of endo- and carboxypeptidases inside the protein bodies and vacuoles in the storage tissue cells of midgermination seeds. Both amino acids and short peptides are transported from the vacuolar compartment into the 619 cytoplasm where amino- and dipeptidases degrade them further. The amino acids are then used to meet metabolic demands in the storage tissue itself, like the biosynthesis of several reserve degrading hydrolases, and to meet the demands imposed by the major site of protein biosynthesis in the germinating seed and growing germling, the embryo axis. Acknowledgements The author expresses his sincere gratitude to Dr C Becker, Dr S Hillmer and above all to Dr D Waddel for critically reading the manuscript which, in addition, has been very much improved by the language editing done by Dr D Waddell. The skilful drawing of the figures by Mrs KJlian and the careful arrangement of the list of references by Mrs. E Bielig are gratefully acknowledged. References Abe Y, Shirane K, Yokosawa H, Matsushita H, Mitta M, Kato I, Ishii S. 1993. Asparaginyl endopeptidases of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis. Journal of Biological Chemistry 268, 3525-9. Adler K, Muntz K. 1983. Origin and development of protein bodies in cotyledons of Vicia faba. Planta 157, 401-10. Akasofu H, Yamauchi D, Minamikawa T. 1990. Nucleotide sequence of the gene for the Vigna mungo sulfhydrylendopeptidase (SH-EP). Nucleic Acids Research 18, 1892. Akasofu H, Yamauchi D, Mitsuhashi W, Minamikawa T. 1989. Nucleotide sequence of cDNA for sulfhydryl-endopeptidase (SH-EP) from cotyledons of germinating Vigna mungo seeds. Nucleic Acids Research 17, 6733. Alpi A, Beevers H. 1981. Effects of leupeptin on proteinase and germination of castor beans. Plant Physiology 68, 851-3. Barrett AJ. 1994. Classification of peptidases. In: Barrett AJ, ed. Proteolytic enzymes: serine and cysteine peptidase. Methods of enzymology, Vol. 244. Abelson JN, MI Simon MI, eds. Academic Press, 1-15. Barton KA, Thompson JF, Madison JT, Rosenthal R, Jarvis NP, Beachy RN. 1982. The biosynthesis and processing of high molecular weight precursors of soybean glycinin subunits. Journal of Biological Chemistry 257, 6089-95. Basha SMM, Beevers L. 1975. The development of proteolytic activity and protein degradation during the germination of Pisum sativum L. Planta (Berl.) 124, 77-87. Bassflner R, Nong VH, Jung R, Saalbach G, Muntz K. 1987. The primary structure of the predominating vicilin storage protein subunit from field bean seeds {Vicia faba L. var. minor cv. Fribo). Nucleic Acids Research 15, 9609. Bassflner R, Wobus U, Rapoport TA. 1984. Signal recognition particle triggers the translation of storage globulin polypeptides from field bean (Vicia faba L.) across mammalian endoplasmic reticulum membrane. FEBS Letters 166, 314-20. Baumgartner B, Chrispeels MJ. 1976. Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans. Plant Physiology 58, 1-6. Baumgartner B, Chrispeels MJ. 1977. Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. European Journal of Biochemistry 77, 223-33. 620 MOntz Baumgartner B, Chrispeels MJ. 1979. Control of protease formation and reserve protein metabolism in the cotyledons of mung bean seedlings. Abhandlungen der Akademie der Wissenschaften der DDR. Abteilung Mathematik, Naturwissenschaften, Technik Akademie-Verlag, Berlin. N4, 115-24. Baumgartner B, Tokuyasu KT, Chrispeels MJ. 1978. Localization of vicihn peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescencc microscopy. Journal of Cell Biology 79, 10-19. Becker C, Fischer J, Nong VH, Mdntz K. 1994. PCR cloning and expression analysis of cDNAs encoding cysteine proteinases from germinating seeds of Vicia saliva L. Plant Molecular Biology 26, 1207-12. Becker C, Shutov AD, Nong VH, Senyuk VI, Jung R, Horstmann C, Fischer J, Nielsen NC, MOntz K. 1995a. Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch {Vicia sativa L.) seeds. European Journal of Biochemistry 228, 456-62. Becker C, Senyuk VI, Shutov AD, Nong VH, Fischer J, Horstmann C, Vaintraub JA, MOntz K. 19956. Proteinase A, a storage globulin degrading endopeptidase of vetch (Vicia sativa L.) seeds, is differentially synthesized in the cotyledons during seed development and germination. Submitted. Belozersky MA, Dunaevsky YE, Voskoboynikova E. 1990. Isolation and properties of a metalloproteinase from buckwheat (Fagopyrum esculentum) seeds. Biochemical Journal 272, 677-82. Bewley JD, Black M. 1994. Seeds, physiology of development and germination, 2nd edn. New York, London: Plenum Press. Bond HM, Bowles DJ. 1983. Characterization of soybean endopeptidase activity using exogenous and endogenous substrates. Plant Physiology 72, 345-50. Bowles DJ, Marcus SE, Pappin DJC, Findlay JBC, Eliopoulus E, Maycox PR, Burgess J. 1986. Post-translational processing of concanavalin A precursors in jackbean cotyledons. Journal of Cell Biology 102, 1284-97. Boylan MT, Sussex IM. 1987. Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons. Planta 170, 343-52. Carrington DM, Auffret A, Hanke DE. 1984. Polypeptide ligation occurs during post-translational modification of concanavalin A. Nature 313, 64-7. Chlan CA, Pyle JB, Legocki AB, Dure III L. 1986. Developmental biochemistry of cotton seed embryogenesis and germination. XVIII. cDNA and amino acid sequences of members of the storage protein families. Plant Molecular Biology 7, 475-89. Chlan CA, Borroto K, Kamalay JA, Dure III L. 1987 Developmental biochemistry of cottonseed embryogenesis and germination. XIX. Sequences and genomic organization of the a-globulin (vicilin) genes of cottonseed. Plant Molecular Biology 9, 533-46. Chrispeels M. 1991. Sorting of proteins in the secretory system. Annual Review of Plant Phvsiologv 42, 21-55. Chrispeels MJ, Baumgartner B. 1978. Trypsin inhibitor in mung bean cotyledons. Purification, characteristics, subcellular localization, and metabolism. Plant Physiology 61, 617-23. Chrispeels MJ, Boulter D. 1975. Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase. Plant Physiology 55, 1031-7. Chrispeels MJ, Raikhel NV. 1992. Short peptide domains target proteins to plant vacuoles. Cell 68, 613-16. Chrispeels MJ, Baumgartner B, Harris N. 1976. Regulation of reserve protein metabolism in the cotyledons of mung bean seedlings. Proceedings of the National Academy of Sciences USA 73, 3168-72. Chrispeels MJ, Higgins TJV, Spencer D. 1982. Assembly of storage protein oligomers in the endoplasmic reticulum and processing of the polypeptides in the protein bodies of developing pea cotyledons. Journal of Cell Biology 93, 306-13. Cornel FA, Plaxton WC. 1994. Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds. Physiologia Plantarum 91, 599-604. Dalbey RE, Heijne G von. 1992. Signal peptidases in prokaryotes and eukaryotes-a new protease family. Trends in Biochemical Sciences 17, 474-8. D'Hondt K, Bosch D, Van Damme J, Goethals M, Vandekerckhove J, Krebbers E. 1993. An aspartic proteinase present in seeds cleaves Arabidopsis 2S albumin precursors in vitro. Journal of Biological Chemistry 268, 20884-91. Diaz P, Wilson KA, Tan-Wilson AL. 1993. Immunocytochemical analysis of proteolysis in germinating soybean. Phytochemistrv 33,961-8. Dickinson CD, Floener LA, Lilley GG, Nielsen NC. 1987. Selfassembly of proglycinin and hybrid proglycinin synthesized in vitro from cDNA. Proceedings of the National Academy of Sciences USA 84, 5225-9. Dickinson CD, Hussein EHA, Nielsen NC. 1989. Role of posttranslational cleavage in glycinin assembly. The Plant Cell 1, 459-69. Doyle JJ, Schuler MA, Godette WD, Zenger V, Beachy R. 1986. The glycosylated seed storage proteins of Glycine max, Phaseolus and Phaseolus vulgaris: structural homologies of genes and proteins. Journal of Biological Chemistry 261, 9228-38. Dunaevsky YE, Belozersky MA. 1989a. Proteolysis of the main storage protein of buckwheat seeds at the early stage of germination. Physiology Plantarum 75, 424-8. Dunaevsky YE, Belozersky MA. 19896. The role of cysteine proteinase and carboxypeptidase in the breakdown of storage proteins in buckwheat seeds. Planta 179, 316-22. Dunaevsky YE, Belozersky MA. 1993. Effects of the embryonic axis and phytohormones on proteolysis of the storage protein in buckwheat seed. Physiologia Plantarum 88, 60—4. Dure IIJ L, Chlan C. 1981. Developmental biochemistry of cotton seed embryogenesis and germination. XII. Purification and properties of principal storage proteins. Plant Physiology 68, 180-6. Elpidina EN, Voskoboynikova NE, Belozersky MA, Dunaevsky YE. 1991. Localization of a metalloproteinase and its inhibitor in the protein bodies of buckwheat seeds. Planta 185, 46-52. Faye L, Chrispeels MJ. 1987. Transport and processing of the glycosylated precursor of Concanavalin A in jack-bean. Planta 170, 217-24. Galili G, Altschuler Y, Levanony H, Giorini-Silfen S, Shimoni Y, Shani N, Karchi H. 1995. Assembly and transport of wheat storage proteins. Journal of Plant Phvsiologv 145, 626-31. Gatehouse JA, Croy RRD, Morton H, Tyler M, Boulter D. 1981. Characterisation and subunit structures of the vicilin storage proteins of pea (Pisum sativum L.). European Journal of Biochemistry 118, 627-33. Gatehouse JA, Lycett GW, Croy RRD, Boulter D. 1982. The post-translational proteolysis of the subunits of vicilin from pea (Pisum sativum L.). Biocltemical Journal 207, 629-32. Gatehouse JA, Lycett GW, Delauney AJ, Croy RRD, Boulter D. 1983. Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.). Biochemical Journal 212, 427-32. Proteolysis of seed storage proteins Gifford DJ, Thakore E, Bewley JD. 1984. Control by the embryo axis of the breakdown of storage proteins in the endosperm of germinated castor bean seed: a role for gibberellic acid. Journal of Experimental Botany 35, 669-77. Guerrero FD, Jones JT, Mullet JE. 1990. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Molecular Biology 15, 11-26. Hara-Nishimura I, Nishimura M. 1987. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons. Plant Physiology 85, 440-5. Hara-Nishimura I, Inoue K, Nishimura M. 1991. A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Utters 294, 89-93. Hara-Nishimura I, Shimida T, Hiraiwa N, Nishimura M. 1995. Vacuolar processing enzyme responsible for maturation of seed proteins. Journal of Plant Physiology 145, 632-40. Hara-Nishimura I, Takeuchi Y, Nishimura M. 1993a. Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni. The Plant CellS, 1651-9. Hara-Nishimura I, Takeuchi Y, Inoue K, Nishimura M. 19936. Vesicle transport and processing of the precursor to 2S albumin in pumpkin. The Plant Journal 4, 793-800. Harley SM, Lord JM. 1985. In vitro endoproteolytic cleavage of castor bean lectin precursor. Plant Science 41, 111-16. Harris N, Chrispeels MJ. 1975. Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physiology 56, 292-9. Heijne G von. 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Research 14, 4683-90. Heim U, Baflmlein H, Wobus U. 1994. The legumin gene family: a reconstructed Vicia faba legumin gene encoding a highmolecular weight subunit is related to type B genes. Plant Molecular Biology 25, 131-5. Heim U, Schubert R, Baumlein H, Wobus U. 1989. The legumin gene family: structure and evolutionary implication of Vicia faba B-type genes and pseudogenes. Plant Molecular Biology 13, 653-63. Herman EM, Melroy DL, Buckout TJ. 1990. Apparent processing of a soybean oil body protein accompanies the onset of oil mobilization. Plant Physiology 94, 341-9. Hiraiwa N, Takeuchi Y, Nishimura M, Hara-Nishimura I. 1993. A vacuolar processing enzyme in maturing and germinating seeds: its distribution and associated changes during development. Plant Cell Physiology 34, 1197-204. Hoh B, Hinz G, Jeong B-K, Robinson DG. 1995. Protein storage vacuoles form de novo during pea cotyledon development. Journal of Cell Science 108, 299-310. Horstmann C, Schlesier B, Otto A, Kostka S, MOntz K. 1993. Polymorphism of legumin subunits from field bean (Vicia faba L. var. minor) and its relation to the corresponding multigene family. Theoretical and Applied Genetics 86, 867-74. Inoue K, Motozaki A, Takeuchi Y, Nishimura M, HaraNishimura I. 1995. Molecular characterization of proteins in protein-body membrane that disappear most rapidly during transformation of protein bodies into vacuoles. The Plant Journal 7, 235-43. Ishii S-I. 1994. Legumain: asparaginyl endopeptidase. Methods in Enzymology 244, 604—15. Jung R, Saalbacfa G, Nielsen NC, MOntz K. 1993. Site-specific limited proteolysis of legumin chJoramphenicol acetyl transferase fusions in vitro and in transgenic tobacco seeds. Journal of Experimental Botany 44, 343-9. 621 Kalinski A, Melroy DL, Dwivedi RS, Herman EM. 1992. A soybean vacuolar protein (P34) related to thiol proteases is synthesized as a glycoprotein precursor during seed maturation. The Journal of Biological Chemistry 267, 12068-76. Kalinski A, Weiseman JM, Matthews BF^ Herman EM. 1990. Molecular cloning of a protein associated with soybean seed oil bodies that is similar to thiol protease of the papain family. The Journal of Biological Chemistry 265, 13843-8. Kembhavi AA, Buttle DJ, Knight CG, Barrett AJ. 1993. The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays. Archives of Biochemistry and Biophysics 303, 208-13. Larkins BA, Hurkman WJ. 1978. Synthesis and deposition of zein in protein bodies of maize endosperm. Plant Physiology 62, 256-63. Lawrence MC, Izard T, Beuchat M, Blagrove RJ, Colman PM. 1994. Structure of phaseolin at 2.2 'A resolution. Implication for a common vicilin/legumin structure and the genetic engineering of seed storage proteins. Journal of Molecular Biology 238, 748-76. Lawrence MC, Suzuki E, Varghese JN, Davis PC, Van Donkelaar A, Tulloch PA, Colman PM. 1990. The three-dimensional structure of the seed storage protein phaseolin at A resolution. EMBO Journal 9, 9-16. Lichtenfeld C, Manteuffel R, MQntz K, Neumann D, Scholz G, Weber E. 1979. Protein degradation and proteolytic activities in germinating field beans (Vicia faba L., var. minor). Biochemie und Physiologie der Pflanzen 174, 255-74. Lichtenfeld C, Manteuffel R, MOntz K, Scholz G. 1981. Protein degradation in germinating legume seeds. Abhandlungen der Akademie der Wissenschaften der DDR, Abteilung Mathematik, Naturwissenschaften, Technik~N4, 133-48. Lively MO, Newsome AL, Nusier M. 1994. Eukaryotic microsomal signal peptidases. In: Barrett AJ, ed. Methods in enzymology. San Diego: Academic Press, Inc., 301-10. Mitsuhashi W, Koshiba T, Minamikawa T. 1986. Separation and characterization of two endopeptidases from cotyledons of germinating Vigna mungo seeds. Plant Physiology 80, 628-34. Mitsuhashi W, Minamikawa T. 1989. Synthesis and posttranslational activation of sulfhydryl-endopeptidase in cotyledons of germinating Vigna mungo seeds. Plant Physiology 89, 274-9. Mflntz K. 1994. Storage and reactivation of high molecular nitrogen compounds in plants. In: Mohr H, Muntz K, eds. The terrestrial nitrogen cycle as influenced by man. Nova Acta Leopoldina N.F. 288, 183-200. MOntz K, Bassuncr R, Lichtenfeld C, Scholz G, Weber E. 1985. Proteolytic cleavage of storage proteins during embryogenesis and germination of legume seeds. Phvsiologie Vegetale 23, 75-94. MOntz K, Horstmann C, Schlesier B. 1986. Seed proteins and their genetics in Vicia faba L. Biologisches Zentralblatt 105, 107-20. MOntz K, Jung R, Saalbach G. 1993. Synthesis, processing, and targeting of legume seed proteins. In: Shewry PR, Stobart K, eds. Seed storage compounds, biosynthesis, interactions, and manipulation. Proceedings of the Phytochemical Society of Europe 5, 128-46. Muramatsu M, Fukazawa C. 1993. A high-order structure of plant storage proprotein allows its second conversion by an asparagine-specific cysteine protease, a novel proteolytic enzyme. European Journal of Biochemistry 215, 123-32. Nandi SK, Palni LMS, Klerk JM de. 1995. The influence of the embryonic axis and cytokinins on reserve mobilization in 622 MClntz germinating lupin seeds. Journal of Experimental Botanv 46, 329-36. Newbigin EJ, De Lumen BO, Chandler PM, Gould A, Blagrove RJ, March JF, Kortt AA, Higgins TJV. 1990. Pea convicilin: structure and primary sequence of the protein and expression of a gene in the seeds of transgenic tobacco. Plania 180,461-70. Ng JD, Ko TP, McPherson A. 1993. Cloning, expression and crystallization of jack bean (Canavaliu ensiformis) canavalin. Plant Physiology 101, 713-28. Nielsen IMC, Jung R, Nam YW, Beaman TW, Oliveira LO, Bassuner R. 1995. Synthesis and assembly of US globulins. Journal of Plant Physiology 145, 641-7. Nielsen SS, Liener IE. 1984. Degradation of the major storage protein of Phaseolus vulgaris during germination. Role of endogenous proteases and protease inhibitors. Plant Physiology 74, 494-8. Nong VH, Becker C, Mttntz K. 1995. cDNA cloning for a putative cysteine proteinase from developing seeds of soybean. Biochimica el Biophysica Ada 1261, 435-8. Okamoto T, Nakayama H, Seta K, Isobe T, Minaraikawa T. 1994. Posttranslational processing of a carboxy-terminal propeptide containing a K.DEL sequence of plant vacuolar cysteine endopeptidase (SH-EP). FEBS Letters 351, 31-4. Osborne TB. 1924. The vegetable proteins. Monographs in biochemistry. London: Longmans, Green & Co. Qi, X, Wilson KA, Tan-Wilson AL. 1992. Characterization of the major protease involved in the soybean £-conglycinin storage protein metabolization. Plant Physiology 99, 725-33. Qi X, Chen R, Wilson KA, Tan-Wilson AL. 1994. Characterization of a soybean /9-conglycinin-degrading protease cleavage site. Plant Physiology 104, 127-33. Rawlings ND, Barrett AJ. 1993. Evolutionary families of peptidases. Biochemical Journal 290, 205-18. Robinson DG, Hinz G. 1995. Multiple mechanism of protein body formation in pea cotyledons. Plant Physiology and Biochemistry (in press). Robinson DG, Hoh B, Hinz G, Jeong BK. 1995. One vacuole or two vacuoles: do protein storage vacuoles arise de novo during pea cotyledon development? Journal of Plant Physiology 145, 654-64. Scholz G, Manteuffel R, MOntz K, Rudolph A. 1983. Low molecular weight polypeptides of vicilin from Vicia faba L. are products of proteolytic breakdown. European Journal of Biochemistry 132, 103-7. Scott MP, Jung R, MOntz K, Nielsen NC. 1992. A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean. Proceedings of the National Academy of Sciences USA 89, 658-62. Shewry PR, Tatham AS. 1990. The prolamin storage proteins of cereal seeds: structure and evolution. Biochemical Journal 267, 1-12. Shimada T, Hiraiwa N, Nishimura M, Hara-Nishimura I. 1994. Vacuolar processing enzyme of soybean that converts proproteins to the corresponding mature forms. Plant Cell Physiology 35,713-18. Shutov AD, Vaintraub IA. 1973. Primary changes of reserve proteins during germination of vetch seeds Fiziologia Rastenji 20, 504-9 (In Russian). Shutov A, Vaintraub IA. 1987. Degradation of storage proteins in germinating seeds. Phytochemistry 26, 1557-66. Shutov AD, Kakhovskaya IA, Braun H, Baumlein H, Muntz K. 1995. Legumin-like and vicilin-like seed storage proteins: evidence for a common single-domain ancestral gene. Journal of Molecular Evolution 41, (in press). Shutov AD, Senyuk VI, Kakhovskaya IA, Pineda J. 1993. High molecular weight products of hydrolysis of soybean glycinin by trypsin. Biokhimiya 58, 301-12. Slightom JL, Drong RF, Klassy RC, Hoffman LM. 1985. Nucleotide sequences from phaseolin cDNA clones: the major storage proteins from Phaseolus vulgaris are encoded by two unique gene families. Nucleic Acids Research 18, 6483-98. Staswick PE. 1990. Novel regulation of vegetative storage protein genes. The Plant Cell 2, 1 - 6 . Staswick PE. 1994. Storage proteins of vegetative plant tissues. Annual Review of Plant Physiology and Plant Molecular Biology 45, 303-22. Takeda O, Miura Y, Mitta M, Matsushita H, Kato I, Abe Y, Yokosawa H, Ishii S. 1994. Isolation and analysis of cDNA encoding a precursor of Canavalia ensiformis asparaginyl endopeptidase (legumain). Journal of Biochemistry 116, 541-6. Tanaka T, Minamikawa T, Yamauchi D, Ogushi Y. 1993 Expression of an endopeptidase (EP-C1) in Phaseolus vulgaris plants. Plant Physiology 101, 421-8. Tanaka T, Yamauchi D, Minamikawa T. 1991. Nucleotide sequence for an endopeptidase (EP-C1) from pods of maturing Phaseolus vulgaris fruits. Plant Molecular Biology 16, 1083^. Thanh VH, Shibasaki K. 1977. Beta-conglycinin from soybean proteins. Isolation and immunological and physico-chemical properties of the monomeric forms. Biochimica et Biophysica Acta 490, 370-84. Vaintraub IA, Kotova LV, Shaha R. 1992. Protein deamidase from germinating wheat grains. FEBS Letters 302, 169-71. Vierstra RD. 1993. Protein degradation in plants. Annual Review of Plant Physiology and Plant Molecular Biology 44, 385-410. Vitale A, Ceriotti A, Denecke J. 1993. The role of the endoplasmic reticulum in protein synthesis, modification and intracellular transport. Journal of Experimental Botany 44, 1417^44. Voelker TA, Herman EM, Chrispeels MJ. 1989. In vitro mutated phytohemaglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability. The Plant Cell 1,95-104. Weschke W, Bassuner R, Nong VH, Czihal A, Baumlein H, Wobus U. 1988. The structure of a Vicia faba vicilin gene. Biochemie und Physiologie der Pflanzen 183, 233—42. Wilson KA. 1986. Role of proteolytic enzymes in the mobilization of protein reserves in the germinating dicot seed. In: Dalling MJ, ed. Plant proteolytic enzymes. Vol. II Boca Raton, Florida: CRC Press Inc., 19-47. Wilson KA, Rightmire BR, Chen JC, Tan-Wilson AL. 1986. Differential proteolysis of glycinin and /3-conglycinin polypeptides during soybean germination and seedling growth. Plant Physiology 82, 71-6. Yamauchi D, Akasofu H, Minamikawa T. 1992. Cysteine endopeptidase from Vigna mungo: gene structure and expressioa Plant Cell Physiology 33, 789-97. Yu WJ, Greenwood JS. 1992. Purification and characterization of a cysteine proteinase involved in globulin hydrolysation in germinated Vicia faba L. Journal of Experimental Botany 45, 261-8.