Sterol regulation in Komagataella phaffii: Identification of sterol regulatory element binding proteins and their activation pathway.

Sterol biosynthesis in eukaryotes is commonly regulated by Sterol Regulatory Element Binding Proteins (SREBPs), membrane-bound transcription factors activated by proteolytic cleavage in response to sterol levels. While extensively studied in mammals, SREBP function and regulation in fungi remain less understood. Here, we identify and characterize two SREBP homologs, Hms1-1 and Hms1-2, in the methylotrophic yeast Komagataella phaffii. Transcriptional and physiological analyses reveal that Hms1-1 and Hms1-2, together with Upc2, form a central regulatory hub controlling sterol-responsive gene expression. Despite largely unchanged total sterol levels, the double deletion mutant (hms1-1Δ hms1-2Δ) exhibits altered expression of sterol biosynthesis genes and increased sensitivity to terbinafine, indicating a sterol-responsive role for SREBPs. Remarkably, K. phaffii retains both Upc2 and SREBP pathways, an unusual dual regulatory system in fungi that highlights evolutionary plasticity. Functional assays demonstrate that overexpression of either HMS1-1 or HMS1-2 rescues the lethality of upc2Δ mutants, revealing redundancy and flexibility in sterol regulation. We show that proteolytic activation of these homologs depends on Scp, a functional analog of mammalian Scap, and the Dsc E3 ligase complex. Comparative analyses identify distinct roles: Hms1-2 acts as the primary, Scp-dependent activator, whereas Hms1-1 is constitutively processed, less Scp-dependent, and likely regulated by post-translational sumoylation. These findings establish K. phaffii as a genetically tractable model for studying SREBP signaling and provide new insights into the evolution and complexity of fungal sterol homeostasis.