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 (SlASAT1-4) sequentially add acyl chains to a sucrose core leading to accumulation of tri and tetraacylated 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 SlASAT1-4 in Nicotiana benthamiana, we were able to pull down a complex consisting of SlASAT1-4, which was confirmed through proteomics. Size exclusion chromatography of the SlASAT pulldown suggests a heteromultimeric complex of around 300 kDa. This study sheds light on the metabolic coordination of acylsugar biosynthesis through formation of a metabolic complex enabling production of chemical defenses.