Functional Characterization of a Signal Peptide Peptidase in Phaffia rhodozyma Reveals a Potential Role in Protein Stress Response but Not in Activation of the SREBP Ortholog Sre1

Sterol regulatory element-binding proteins (SREBPs) regulate lipid homeostasis and coordinate sterol metabolism and carotenogenesis in the astaxanthin-producing yeast <i>Phaffia rhodozyma</i>. While Sre1, the SREBP ortholog, and the site-2 protease Stp1 have been identified as essential components of this pathway in <i>P. rhodozyma</i>, additional factors involved in Sre1 processing or regulation remain unknown. In <i>Aspergillus</i> species, a signal peptide peptidase contributes to the activation of the SREBP ortholog, raising the possibility of a similar role in this yeast. In this work, we identified and characterized the <i>P. rhodozyma</i> signal peptide peptidase (SppA) homolog. Sequence analysis, domain prediction, and phylogenetic analyses supported its classification within the SPP family of intramembrane aspartyl proteases. To evaluate its functional role, Δ<i>sppA</i> mutants were constructed in genetic backgrounds with constitutive Sre1 activity, including the <i>cyp61</i>^- mutant and a strain expressing the active form of Sre1 (Sre1N). Deletion of <i>SPPA</i> did not alter sensitivity to clotrimazole or cobalt chloride, nor affect pigmentation, indicating that SppA is not required for Sre1 activation in <i>P. rhodozyma</i>. Transcriptomic analyses further showed that expression of <i>SRE1</i> and of its known target genes remained unchanged upon <i>SPPA</i> deletion. Interestingly, the loss of SppA in the Sre1N background caused marked downregulation of genes associated with protein refolding and unfolded protein binding. In agreement with these transcriptional changes, the Sre1NΔ<i>sppA</i> strain displayed increased sensitivity to dithiothreitol. These findings suggest that, although SppA is not involved in Sre1 activation in <i>P. rhodozyma</i>, it may play a role in protein stress-related processes. Future studies will be required to define the molecular mechanisms underlying this role and its integration with protein homeostasis networks.