A metabolic complex of acyltransferase enzymes involved in tomato acylsugar biosynthesis

Specialized metabolites mediate diverse plant-environment interactions. Recent work has begun to enzymatically characterize entire plant specialized metabolic pathways; however, little is known about how different pathway components organize and interact within the cell. Here we use acylsugars-a class of specialized metabolites-to explore metabolic complex formation. In Solanum lycopersicum (tomato), four trichome-localized acylsugar acyltransferases (SlASAT1- SlASAT4) sequentially add acyl chains to a sucrose core leading to accumulation of tri- and tetra-acylated sucroses. Confocal microscopy demonstrates that tomato ASATs localize to distinct subcellular locations, including the mitochondria, cytosol, and endoplasmic reticulum. To explore pairwise protein-protein interactions in acylsugar biosynthesis, we used various techniques relying on different interaction principles, including co-immunoprecipitation, split-luciferase assays, and bimolecular fluorescence complementation, all demonstrating pairwise SlASAT interactions. Following transient expression of SlASAT1-SlASAT4 in Nicotiana benthamiana, we were able to pull down a complex consisting of SlASAT1-SlASAT4, which was confirmed through proteomics. Size exclusion chromatography of the SlASAT pulldown suggests a heteromultimeric complex of ∼300 kDa. This study sheds light on the metabolic coordination of acylsugar biosynthesis through formation of a metabolic complex enabling production of chemical defenses.