Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters.

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.

Auxin-transporting ABC transporters are defined by a conserved D/E-P motif regulated by a prolylisomerase.

The plant hormone auxin must be transported throughout plants in a cell-to-cell manner to affect its various physiological functions. ABCB transporters are critical for this polar auxin distribution, but the regulatory mechanisms controlling their function is not fully understood. The auxin transport activity of ABCB1 was suggested to be regulated by a physical interaction with FKBP42/Twisted Dwarf1 (TWD1), a peptidylprolyl cis-trans isomerase (PPIase), but all attempts to demonstrate such a PPIase activity by TWD1 have failed so far. By using a structure-based approach, we identified several surface-exposed proline residues in the nucleotide binding domain and linker of Arabidopsis ABCB1, mutations of which do not alter ABCB1 protein stability or location but do affect its transport activity. P1008 is part of a conserved signature D/E-P motif that seems to be specific for auxin-transporting ABCBs, which we now refer to as ATAs. Mutation of the acidic residue also abolishes auxin transport activity by ABCB1. All higher plant ABCBs for which auxin transport has been conclusively proven carry this conserved motif, underlining its predictive potential. Introduction of this D/E-P motif into malate importer, ABCB14, increases both its malate and its background auxin transport activity, suggesting that this motif has an impact on transport capacity. The D/E-P1008 motif is also important for ABCB1-TWD1 interactions and activation of ABCB1-mediated auxin transport by TWD1. In summary, our data imply a new function for TWD1 acting as a putative activator of ABCB-mediated auxin transport by cis-trans isomerization of peptidyl-prolyl bonds.

An LRR receptor kinase controls ABC transporter substrate preferences during plant growth-defense decisions.

The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions.