Polar Localization of the Borate Exporter BOR1 Requires AP2-Dependent Endocytosis

Abstract

Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking mor s-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxF sorting motifs, which are in a cytosolic loop of BOR1. A yeast twohybrid assay supported the interaction of the AP2 complex m-subunit with the C-terminal tail but not with the YxxF motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

Cell polarity: Regulators and mechanisms in plants

“…In addition, both boron (B) transporters NIP5;1 and BOR1 require the AP2‐controlled CME pathway to maintain their polarization at the PM (Yoshinari et al 2016; Wang et al 2017a). Interestingly, the BOR1 protein associates with the AP2 complex and under the low‐B conditions, the AP2‐dependent CME maintains the polar localization of BOR1 to support plant growth and face the challenge of boron deficiency (Yoshinari et al 2019).…”

Two Pathways for Trafficking the Borate Receptor BOR1

“…In their recent report, Yoshinari et al (2019) explore the separate endocytic pathways regulating BOR1 localization to the plasma membrane and degradation in the vacuole. In the trafficking of BOR1, recycling between the plasma membrane and trans-Golgi network/ early endosomes maintains the polar localization of BOR1, and a degradation pathway from the plasma membrane to multivesicular bodies/late endosomes helps BOR1 make its way to the vacuole.…”

“…In the trafficking of BOR1, recycling between the plasma membrane and trans-Golgi network/ early endosomes maintains the polar localization of BOR1, and a degradation pathway from the plasma membrane to multivesicular bodies/late endosomes helps BOR1 make its way to the vacuole. The main question asked by Yoshinari et al (2019) is what pathways mediate the polar localization and vacuolar sorting of BOR1? As shown in this report, the two pathways that manage BOR1 membrane localization include one that relies on the clathrin adaptor protein 2 (AP2) for BOR1 polarization at the plasma membrane and a clathrin/ AP2-independent pathway that functions in BOR1 degradation.…”

“…In plants, loss-of-function mutants of AP2 subunits have pleiotropic effects at the cellular and physiological levels, most likely because of the misregulation of the plasma membrane proteins that they help transport/localize (Fan et al, 2013;Yamaoka et al, 2013). To separate the endocytic pathways involved in BOR1 polar localization and degradation, Yoshinari et al (2019) used BOR1-GFP constructs expressed in ap2m mutants and observed a disrupted localization pattern and a reduced level of internalization from the plasma membrane compared with the wild type. However, BOR1 endocytosis initiated by high-boron conditions was not impaired in ap2m mutants, suggesting that the degradation pathway of BOR1 is AP2 independent.…”

“…The BOR1 and PIN proteins include a YXXF motif, but interestingly, Yoshinari et al (2019) show that altering this motif does not affect BOR1-AP2 interactions; rather, these interactions occur through domains residing on the C-terminal tail, suggesting that BOR1 Figure 1. Separation of AP2-dependent and -independent endocytic trafficking of BOR1.…”