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 the 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 (AtPHT1;1) in the tobacco relative Nicotiana benthamiana transient expression system caused a portion of AtPHF1 to distribute into AtSAR1b- and AtSEC24a-labelled ER exit sites (ERES). We demonstrated that AtPHF1 interacts with AtSAR1b and AtSAR1c based on tripartite split-GFP association in agro-infiltrated N. benthamiana leaves and verified this interaction using miniTurbo-based proximity labelling. We also confirmed its physiological relevance by co-immunoprecipitating endogenous AtPHF1 with AtSAR1c-GFP in Arabidopsis transgenic lines. Importantly, AtPHF1 preferentially interacts with GDP-locked AtSAR1. Therefore, we propose that AtPHF1 or the AtPHT1;1-AtPHF1 complex interacts with SAR1 GTPase to participate in the early step of COPII assembly for the ER export of PHT1 transporters.