Increased phosphate transport of Arabidopsis thalianaPht1;1 by site-directed mutagenesis of tyrosine 312 may be attributed to the disruption of homomeric interactions

Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in plantaPi translocation to maintain optimal growth and development. The study of the specificities and biochemical properties of Pht1 transporters will contribute to improving the current understanding of plant phosphorus homeostasis and use-efficiency. In this study, we show through split in vivo interaction methods and in vitro analysis of microsomal root tissues that Arabidopsis thalianaPht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1Y312D, Pht1;1Y312A and Pht1;1Y312F, was used to analyse the role of a putative Pi binding residue (Tyr 312) in Pht1;1 transporter oligomerization and function. The homomeric interaction among Pht1;1 proteins was disrupted by mutation of Tyr 312 to Asp, but not to Ala or Phe. In addition, the Pht1;1Y312D variant conferred enhanced Pi transport when expressed in yeast cells. In contrast, mutation of Tyr 312 to Ala or Phe did not affect Pht1;1 transport kinetics. Our study demonstrates that modifications to the Pht1;1 higher-order structure affects Pi transport, suggesting that oligomerization may serve as a regulatory mechanism for modulating Pi uptake. Oligomerization has been shown to be an important aspect of regulation and function for some membrane transporters. Herein we demonstrate that the Arabidopsis Pht1;1 and Pht1;4 phosphate transporters form homomeric and heteromeric oligomers. Mutation of a tyrosine residue abolished homo-oligomerization of Pht1;1 and also conferred enhanced phosphate transport when expressed in yeast. The results suggest an active site-oligomerization relationship in which oligomerization serves as a mechanism to regulate transporter activity.