The leucine zipper domain of the transcriptional repressor Opi1 underlies a signal transduction mechanism regulating lipid synthesis

In Saccharomyces cerevisiae the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid (PL) synthesis responding to the abundance of the PL precursor phosphatidic acid (PA) at the endoplasmic reticulum (ER). We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function (GOF) mutations and the characterization of the strongest variant identified, Opi1. LZ modelling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1 variant is dominant, and its phenotype is attenuated when loss of function (LOF) mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region we performed in vitro analyses to explore inter-domain interactions. An Opi1 probe interacted with Opi1, the C-terminus that bears the Activator Interacting Domain (AID). LZ or AID LOF mutations attenuated the interaction of the probes, but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.