Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signaling initiation, which requires binding of a BR molecule to the extracellular domains of the plasma membrane-localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter ABCB19 functions as a BR exporter and, together with its close homolog ABCB1, positively regulates BR signaling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 can be stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 were determined in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states. In the BL-bound structure, BL is bound to the hydrophobic cavity formed by the transmembrane domain and triggers local conformational changes. Together, our data provide additional insights into ABC transporter-mediated BR export.

Department of Organic and Macromolecular Chemistry Ghent University 9000 Ghent Belgium

Institute of Science and Technology Austria Am Campus 1 3400 Klosterneuburg Austria

Laboratory of Growth Regulators Institute of Experimental Botany The Czech Academy of Sciences and Palacký University 77900 Olomouc Czech Republic

MOE Key Laboratory for Membraneless Organelles and Cellular Dynamics Hefei National Research Center for Physical Sciences at the Microscale Center for Advanced Interdisciplinary Science and Biomedicine of IHM Division of Life Sciences and Medicine University of Science and Technology of China Hefei 230027 China

University Department of Plant Biotechnology and Bioinformatics Ghent University 9052 Ghent Belgium; Center for Plant Systems Biology VIB 9052 Ghent Belgium

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