The 11S globulins are seed proteins found in many grain legumes. After synthesis on endoplasmic reticulum, the globulins are assembled during a series of steps that involve post-translational events and transport of precursors through several intracellular compartments. As an aid in dissecting this process, an in vitro synthesis and assembly system was developed that results in the formation of trimers equivalent in size to oligomers found in endoplasmic reticulum. Assembly of proglycinin trimers in vitro is dependent upon ATP. This observation, together with other evidence, is consistent with the hypothesis that molecular chaperones are involved in the assembly of proglobulin trimers. The 11S proglobulin subunits in trimers are cleaved post-translationally at an ASN-GLY bond that has been conserved during evolution. Evidence from studies carried out both in vivo and in vitro establish that cleavage of this bond is prerequisite for further assembly of trimers into hexamers. Proteolytic activity has been detected in developing seeds that is capable of cleaving the conserved ASN-GLY. The preponderance of this proteolytic activity in developing seeds is due to glycosylated proteins (Scott et al., 1992), although a small amount of the total activity appears associated with a non-glycosylated peptide. To study the specificity of the proteases that are glycosylated, mutant proglobulin subunits or peptides were constructed in which amino acids around the conserved ASN-GLY bond were modified, and then the modified subunits were assembled into trimers and subjected to proteolysis. Deletion of the asparagine on the P1 side of the cleaved peptide bond, or substitution of aspartate or glutamine for the asparagine, eliminated cleavage of the subunit precursor. Conservative changes of the amino acids on the COOH-terminal side of the cleavage site (P1' to P7' positions) seemed not to hinder cleavage of proglobulin into mature acidic and basic polypeptides. In contrast to the normal proglycinin subunits found in trimers, unmodified monomers, unfolded or misfolded normal. 11S proglobulins, fusions of legumin polypeptides with CAT, and several high methionine mutants of proglycinin, are cleaved into small peptide fragments by the purified, glycosylated protease. Our results demonstrate that cleavage sites other than the conserved ASN-GLY bond found in unmodified proglobulin subunits are inaccessible to the protease when they are contained in trimers.
