Proximity Labelling Confirms the Involvement of Papain-Like Cysteine Proteases and Chaperones in Cyclotide Biosynthesis

Abstract

AbstractCyclotides are disulfide-rich insecticidal peptides from plants that harbour a cyclic cystine knot motif that imparts them with high stability. Like most ribosomally encoded peptides, cyclotides are produced as precursor proteins that are subsequently processed to maturity through proteolytic activity. Although the final maturation of the prototypic cyclotide kalata B1 (kB1) is known to involve asparaginyl endopeptidases, the role of the in planta folding machinery is less well characterized, as is also the case for in planta N-terminal processing enzyme(s). Here, we used proximity labelling to identify proteins involved in the biosynthesis of kB1 in both a cyclotide-bearing plant species, Petunia × hybrida [Regel], and in Nicotiana benthamiana [Domin] that does not naturally produce cyclotides. Together, several ER resident chaperones, protein disulfide isomerases, a papain-like cysteine protease, and an asparaginyl endopeptidase were identified in the kB1 interactome. We found that overexpression of the identified papain-like cysteine protease resulted in an improvement in the yield of cyclic kB1, while this was not the case for the overexpression of protein disulfide isomerases, despite their interaction being verified using bimolecular fluorescence complementation. Together, these results provide a significant advance in our understanding of the role of auxiliary biosynthetic elements for heterologous cyclotide production in plants.