Mechanistic insight into substrate specificity of plant glucosinolate transporters

Abstract

Plants depend on transport processes for correct allocation of specialized metabolites. This is important for optimal defense, avoidance of autotoxicity, connecting compartmented biosynthetic modules and more. Transport of a wide variety of specialized metabolites is mediated by transporters from the Nitrate and Peptide transporter Family (NPF), which belongs to the Major Facilitator Superfamily (MFS). However, the mechanism by which NPF members recognize and transport specialized metabolites remains unknown. Here we mutate eight residues to reciprocally swap the substrate-preference of two closely related glucosinolate transporters (GTRs). Seven of these residues assemble in a ring-like structure in all conformations of the transporters. We labeled the ring-like structure a selectivity filter and based on docking studies, we propose that the interaction between the selectivity filter and the glucosinolate side chain determines whether a given glucosinolate is recognized as a substrate. Besides partly explaining the distinct substrate preference of GTR1 (NPF2.10) and GTR3 (NPF2.9), this study proposes fundamental principles of substrate recognition in the NPF and establishes the GTR subclade as a novel model system for studying structure function relationships in the NPF.

Identification of key amino acid residues in AtUMAMIT29 for transport of glucosinolates

“…We do, however, not observe a clear spatially distinguished subdomain for the five key residues compared with the remaining six residues. This is different from the glucosinolate importer NPF2.9 and NPF2.10 of which the key residues involved in glucosinolate selectivity form a ring structure ( Kanstrup et al., 2023 ). This suggests that the identified key residues within the cavity in UMAMIT29 play a role in different steps of the transport cycle.…”