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

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 instead specifically assists the ER exit of the plant PHOSPHATE TRANSPORTER1 (PHT1) family. However, the underlying molecular mechanism remains to be elucidated. In this study, we showed that the overexpression of Arabidopsis thaliana PHT1;1 ( At PHT1;1) but not of SUGAR TRANSPORTER 1 ( At STP1) caused a portion of At PHF1 distribution into At SAR1b-, At SAR1c- and At SEC24a-labeled ER exit sites in the tobacco transient expression system. Based on the in-planta tripartite split-GFP association, At PHF1 interacted with At SAR1b and At SAR1c but not with other COPII-related proteins. By performing the miniTurbo-based proximity labeling in agro-infiltrated tobacco leaves, we verified the interaction of At PHF1 and At SAR1b and demonstrated its physiological relevance by co-immunoprecipitation of the endogenous At PHF1 and At PHT1;1/2/3 proteins with At SAR1c-GFP using Arabidopsis transgenic lines. Furthermore, while both the cytosolic and transmembrane domains of At PHF1 contribute to the interaction with At SAR1b and At SAR1c, At PHF1 preferentially interacted with the GDP-locked inactive form of At SAR1b. On the basis of these findings, we propose that by interacting with SAR1 GTPase, PHF1 participates in the early step of COPII recruitment for the ER export of PHT1 proteins.