Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling. Editor’s summary The plant ABC transporter ABCB19 has long been implicated in auxin transport. Ying et al. found an alternative role for ABCB19 as a transporter of another hormone group, brassinosteroids. The authors determined the cryoelectron microscopy structure and found a hydrophobic pocket where brassinolides can bind. They also demonstrated that brassinosteroids triggered ABCB19 ATP hydrolase activity and that mutating specific amino acids in the ABCB19 protein disrupted brassinosteroid transport. The work challenges the auxin-focused view of ABCB19 function and provides an explanation for how brassinosteroids might be exported out of cells to the site of their perception. —Madeleine Seale Biochemical, in planta, and structural analyses demonstrate that ABCB19 is a brassinosteroid exporter. INTRODUCTION Brassinosteroids are steroidal phytohormones essential for plant growth and development and for response to environmental stresses. The brassinosteroid signaling pathway has been well studied, and key membrane-localized receptors, co-receptors, and downstream factors have been identified and characterized. Brassinosteroids are synthesized in the cell interior but perceived in the apoplast through binding to the extracellular part of the plasma membrane–localized receptor complex, which initiates the signaling pathway. As polar molecules, the bioactive brassinosteroids might not easily permeate the cell membrane through simple diffusion. The mechanism by which the intracellularly synthesized brassinosteroids are exported remains elusive. The Arabidopsis ATP-binding cassette (ABC) transporter ABCB19 has been proposed to play a role in the distribution and export of another classical phytohormone, auxin, but its substrate specificity awaits thorough investigation. RATIONALE In this work, we tested the ATPase activities of the Arabidopsis ABCB19 in the presence of different phytohormones, including auxins and brassinosteroids. Next, the binding affinities between ABCB19 and bioactive brassinosteroids were determined. In vitro proteoliposome-based and in vivo protoplast-based assays were used to evaluate the transport of brassinosteroids by ABCB19. Structural analyses were performed for the wild-type ABCB19 and the ATPase inactive mutant in the ATP-bound state in the absence or presence of brassinosteroids. Genetic analyses were performed to confirm the role of ABCB19 and its close homolog, ABCB1, in brassinosteroid signaling. RESULTS The ATPase activity of ABCB19 can be stimulated by the bioactive brassinosteroids in a dose-dependent manner, but not by auxins or the brassinosteroid biosynthesis precursors. The binding affinities between ABCB19 and the bioactive brassinosteroids are at micromolar range, as determined by isothermal titration calorimetry. Proteoliposome-based and Arabidopsis protoplast–based transport assays both proved that ABCB19 can transport brassinosteroids. The structures of ABCB19 revealed a typical architecture of the type IV ABC transporters, consisting of two transmembrane domains and two nucleotide-binding domains. An extra regulatory domain was identified between the transmembrane domains and may play an inhibitory role in ABCB19 activity regulation. The brassinosteroid molecule binds to an intracellularly facing cavity in the transmembrane region of ABCB19 through both hydrophobic interactions and hydrogen bonds. The abcb1 abcb19 double mutant resembled the brassinosteroid biosynthetic mutants with decreased brassinosteroid signaling and was insensitive to the application of either brassinolide or the biosynthetic precursor 22-hydroxycampesterol. By contrast, plants overexpressing ABCB1 or ABCB19 show increased brassinosteroid signaling. Moreover, the plasma membrane abundance of ABCB1 and ABCB19 was regulated by brassinosteroids. CONCLUSION In vivo and in vitro analyses collectively show that the Arabidopsis ABC transporter ABCB19 functions as a brassinosteroid exporter and positively regulates brassinosteroid signaling together with ABCB1. The determined structures provide insights into the molecular architecture of ABCB19 and how it recognizes the brassinosteroid molecule. These results provide a basis for further investigations on the control of ABCB19 transport activity by phosphorylation of its regulatory domain, and pave the way for future studies of both auxin and brassinosteroid transport and their signaling cross-talk in plants. Model of brassinosteroid export and signaling in plants. Brassinosteroids are synthesized in the cell interior, likely in the endoplasmic reticulum, where most of the biosynthetic enzymes are localized. The Arabidopsis ABCB19 functions as a brassinosteroid exporter utilizing the energy of ATP hydrolysis. Brassinosteroids are perceived by the plasma membrane–localized receptors BRI1 and BAK1, which then trigger downstream signaling. BR, brassinosteroid; BRI1, BR INSENSITIVE1; BAK1, BRI1-ASSOCIATED KINASE1.