Increased phosphate transport of Arabidopsis thaliana Pht1;1 by site‐directed mutagenesis of tyrosine 312 may be attributed to the disruption of homomeric interactions

Abstract

Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in planta Pi 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 thaliana Pht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1(Y312D), Pht1;1(Y312A) and Pht1;1(Y312F), 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;1(Y312D) 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.

A vacuolar phosphate transporter essential for phosphate homeostasis inArabidopsis

“…These include the phosphate transporters (PHTs) and PHO-type Pi transporters. The plasma membrane-located PHT1s are responsible for the absorption of phosphate, as has been studied in Arabidopsis and rice (3,4,24,25). For Pi translocation, the Golgi located PHO1 plays an important role (1,16).…”

The Journey of Arsenic from Soil to Grain in Rice

“…Till date a number of phosphate transporters have been identified for the As(V) uptake in different plants. Arsenate enters into the cell via OsPHT1;1 (Kamiya et al, 2013), OsPHT1;8 (Wu Z. et al 2011) in rice and AtPHT1;1, AtPHT1;4, AtPHT1;5, AtPHT1;7, AtPHT1;8, AtPHT1;9 in A. thaliana (Shin et al, 2004; Catarecha et al, 2007; Remy et al, 2012; LeBlanc et al, 2013; Fontenot et al, 2015). Wang et al (2016) compared As(V) tolerance of aus variety Kasalath with japonica variety Nipponbare and found Kasalath to be more tolerant to As(V) than Nipponbare.…”

Techniques for the analysis of protein-protein interactions in vivo

“…The split-ubiquitin system has been used extensively in plant research to identify interactions of membrane proteins such as G-proteins (Aranda-Sicilia et al, 2015), phosphate transporters (Fontenot et al, 2015), and aquaporins (Besserer et al, 2012;Hachez et al, 2014), potassium channels (Obrdlik et al, 2004), and their interactions with SNARE proteins (Honsbein et al, 2009;Grefen et al, 2015;Zhang et al, 2015), specifically using the vectors modified by Petr Obrdlik and coworkers (Obrdlik et al, 2004). Improvements to the original vector system included increasing the signal-to-noise ratio through introducing a Met-repressible promoter driving the bait Cub fusion (Obrdlik et al, 2004), incorporating cloning sites for in vivo cloning as well as Gateway recombination technology (Obrdlik et al, 2004;Grefen et al, 2007Grefen et al, , 2009, and altering linkers and tags (Grefen and Blatt, 2012b), all of which, together with the development of strains that allow a mating-based approach to be taken (Ludewig et al, 2003), have made the system suitable for high-throughput analysis (Box I; Lalonde et al, 2010;Chen et al, 2012b;Jones et al, 2014).…”