Dual transcriptional and post-transcriptional regulation by the bHLH proteins Rtg1 and RtgX in Komagataella phaffii

In Saccharomyces cerevisiae, the basic helix-loop-helix (bHLH) transcription factors Rtg1 and Rtg3 mediate retrograde signaling. In Candida albicans, the Rtg1-Rtg3 complex regulates galactose and sphingolipid metabolism and contributes to virulence. In the methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris), Rtg1 (KpRtg1) controls glutamate and methanol metabolism by regulating the synthesis of GDH2 (glutamate dehydrogenase 2), PEPCK (phosphoenolpyruvate carboxykinase), and AOX1 (alcohol oxidase 1). The function of the putative Rtg3 ortholog, designated here as KpRtgX, has remained unknown. In this study, we identify KpRtgX as the missing regulatory partner of KpRtg1. Deletion of either KpRTG1 or KpRTGX produces identical phenotypes, suggesting functional overlap. KpRtgX localizes to both cytosol and nucleus, activates AOX1 transcription in the nucleus, and regulates GDH2 and PEPCK post-transcriptionally in the cytosol. Notably, KpRtgX protein but not mRNA is strongly reduced in ΔKprtg1 cells, indicating that KpRtg1 stabilizes KpRtgX. The two proteins interact through their bHLH domains when expressed in K. phaffii but not in Escherichia coli. Mutation of conserved arginine residues in the KpRtgX bHLH domain reduces its expression, implicating these residues in complex formation with KpRtg1. KpRtg1-KpRtgX complex is required both for GDH2 protein stability and for 5' UTR-dependent translation of PEPCK mRNA during glutamate utilization.