Proteases of vacuoles and other endomembrane compartments possibly involve proteolytic processing of soybean β-conglycinin subunits.

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conclusionGmCP3 in soybean seed vacuoles is a candidate processing enzyme of β-conglycinin subunits. However, a different type of proteases may be involved in unconventional secretion of β-conglycinin propeptides. Soybean β-conglycinin subunits, a 7S vicilin class of seed storage proteins (SSPs) of seed vacuoles, are subject to proteolytic cleavage of the N-terminal propeptide for subunit maturation. Unlike other SSPs, this process is independent of asparagine residues. It occurs at the C-terminal of lysine residues, indicating the involvement of unidentified processing enzymes outside the vacuolar processing enzyme (VPE) family of proteases. In this study, we hypothesized that GmCP3, a seed-specific vacuolar protease belonging to the papain-like cysteine protease (PLCP) family, might be responsible for this process and analyzed the relationship between its accumulation and processing. The results showed that GmCP3 accumulated abundantly during the synthesis of β-conglycinin subunits and significantly decreased once the processing was complete. Immunoelectron microscopy of developing seed cells showed the accumulation of both, GmCP3 and β-conglycinin propeptides, in vacuolar-associated compartments, such as protein storage vacuoles (PSVs) and prevacuolar compartments (PVCs). Unexpectedly, labeling specific of the propeptide region of β-conglycinin subunits, but not the mature subunit region, was observed in extracellular regions. These results indicate that GmCP3 is a possible candidate β-conglycinin processing enzyme of vacuoles, though other proteases may have a role in the secretion of propeptides.