The Arabidopsis PHOSPHATE 1 exporter undergoes constitutive internalization via clathrin-mediated endocytosis

Inorganic phosphate (Pi) homeostasis is essential for plant growth and depends on the transport of Pi across cells. In Arabidopsis thaliana, PHOSPHATE 1 (PHO1) is present in the root pericycle and xylem parenchyma where it exports Pi into the xylem apoplast for its transfer to shoots. PHO1 consists of a cytosolic SPX domain followed by membrane-spanning alpha-helices and ends with the EXS domain, which participates in the steady-state localization of PHO1 to the Golgi and trans-Golgi network (TGN). However, PHO1 exports Pi across the plasma membrane (PM), making its localization difficult to reconcile with its function. To investigate whether PHO1 transiently associates with the PM, we inhibited clathrin-mediated endocytosis (CME) by overexpressing AUXILIN-LIKE 2 or HUB1. Inhibiting CME resulted in PHO1 re-localization from the Golgi/TGN to the PM when PHO1 was expressed in Arabidopsis root pericycle or epidermis or Nicotiana benthamiana leaf epidermal cells. A fusion protein between the PHO1 EXS region and GFP was stabilized at the PM by CME inhibition, indicating that the EXS domain plays an important role in sorting PHO1 to/from the PM. PHO1 internalization from the PM occurred independently of AP2 and was not influenced by Pi deficiency, the ubiquitin-conjugating E2 PHO2, or the potential ubiquitination of cytosolic lysines in the EXS domain. PM-stabilized PHO1 showed reduced root-to-shoot Pi export activity, indicating that CME of PHO1 may be important for its optimal Pi export activity and plant Pi homeostasis.