The sequence of cDNA coding for a sulphur-rich storage protein from Lupinus angustifolius L., conglutin gamma, was determined. The coding region contained an N-terminal leader peptide of 28 amino acids which directly preceded subunits of M(r) 28,239 and 16,517. Extensive sequence homology between the protein encoded by conglutin gamma cDNA and basic 7S globulin from soybean was observed. Sequence homology to proteins from other classes of storage proteins, 11S, 7S and 2S, was limited to short and highly fragmented sequences. The amino acid sequence, Asn-Gly-Leu-Glu-Glu-Thr, characteristic of the primary site for post-translational cleavage of the precursors of 11S proteins, was absent from the sequence predicted for prepro-conglutin gamma. It is concluded that conglutin gamma is a representative of a fourth type of storage protein in legumes, distinct from the 11S, 7S and 2S storage protein families.

The biosynthesis of conglutin delta has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin delta formed the major sink for [35S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin delta was determined. The structure of the precursor polypeptide for conglutin delta predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of Mr 4520, together with a linking region of 13 amino acids and a subunit polypeptide of Mr 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin delta. Comparison of the sequences of conglutin delta with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin delta, were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.

Using Nycodenz, a novel density gradient medium, we isolated intact protein bodies from developing seeds of Lupinus angustifolius L. (cultivar Unicrop) and achieved excellent separation from the endoplasmic reticulum, mitochondria, and other organelles.The distribution of the storage protein conglutin-,# was taken as evidence that up to 96% of the protein bodies remained intact on the gradients and banded at 1.25 grams per milliliter. The protein bodies also contained the three other abundant proteins present in L. angustifolius seeds: conglutins-a, -y, and -a. Pulse labeling experiments were carried out to determine the site of proteolytic processing of conglutin-a, a legumin-like 1 lSvedberg unit storage protein. Cotyledons aged either 33 or 40 days after flowering were pulsed with [3H]leucine. Protein bodies obtained from the cotyledons aged 33 days after flowering contained only the labeled precursors of conglutin-a with molecular weights 85,000, 72,000, and 64,000, even after a 4 hour chase of the radioactivity. Protein bodies obtained from the cotyledons aged 40 days after flowering contained the same radioactive precursors if the tissue had been pulsed for 2 hours, and the processing products of these precursors when the tissue had been chased for 4 hours. These studies confirm that the subcellular location of proteolytic cleavage of this legumin-like protein is the protein body, that this activity is detected only in protein bodies from lupin seeds aged between 33 and 40 days of seed development after flowering and that protein bodies from seeds younger than this contain only unprocessed conglutin-a.Seeds of the grain legume Lupinus angustifolius contain three predominant storage proteins and one relatively minor storage protein. The predominant proteins, conglutins-a, -3, and -6, are members of the widespread lIS, 7S, and 2S families of storage proteins, respectively. The other protein in seeds of lupins, conglutin-y, appears to be an example of a fourth type of storage protein (3). Conglutin-y is a methionine and cysteine-rich protein and has physical and chemical properties quite distinct from the other classes of storage proteins (5, 25). Conglutin-y in lupins and the homologous relative in soybeans, "basic 7S globulin" (17) are both present only in relatively small amounts in seeds of the respective species.

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