Identification of Golgin Imh1 phosphorylation residues under ER stress in yeast

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which acts through various mechanisms to reduce ER stress. We previously found ER stress induced by tunicamycin (TM) treatment promotes the activation of small GTPase Arl1 and thereby increasing the recruitment of downstream effector golgin Imh1 to the late-Golgi. However, the role of Imh1 under ER stress remains unknown. In this study, we found Imh1 is required for the recycling of two SNAREs, Snc1 and Tlg1, upon TM-induced ER stress. Due to Imh1 is already known as phosphorylation protein, we wonder whether phosphorylation play roles in regulating the function of Imh1 under ER stress. We utilized SILAC method to identify the phosphorylation sites on Imh1 upon TM treatment through MS analysis and found several sites increased phosphorylation under ER stress. We finally selected the predominant S25/T27 phosphorylated residues and study the mechanism of how S25/T27 phosphorylation regulates the function of Imh1 in SNARE transport upon TM treatment.