Involvement of PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 in COPII assembly by interacting with SAR1 GTPase

Inorganic phosphate (Pi) uptake and translocation are crucial for plant growth and development, relying on plasma membrane targeting of PHOSPHATE TRANSPORTER1 (PHT1) transporters. The plant-specific endoplasmic reticulum (ER)-resident PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) is structurally related to SEC12, which initiates the coat protein complex II (COPII) assembly as a guanine nucleotide exchange factor (GEF) by activating the small GTPase SAR1. In contrast, PHF1 loses the conserved catalytic residues critical for GEF activity and specifically assists the ER exit of the PHT1 transporters. However, the underlying molecular mechanism remains unknown. In this study, we showed that overexpression of Arabidopsis thaliana PHT1;1 ( At PHT1;1) in the tobacco transient expression system caused a portion of At PHF1 distribution into At SAR1b- and At SEC24a-labeled ER exit sites. We demonstrated that At PHF1 interacts with At SAR1b and At SAR1c based on the tripartite split-GFP association in agro-infiltrated tobacco leaves and verified this interaction using miniTurbo-based proximity labeling. We also confirmed its physiological relevance by co-immunoprecipitation of the endogenous At PHF1 with At SAR1c-GFP in Arabidopsis transgenic lines. Importantly, At PHF1 preferentially interacts with the GDP-locked At SAR1. Therefore, we propose that PHF1 interacts with the SAR1 GTPase to participate in the early step of COPII recruitment for the ER export of PHT1 transporters.