Paper ID: 300
1 Stockholm University (Sweden)
2 Stockholm University (San Marino)
The Ssy1-Ptr3-Ssy5 (SPS) signaling pathway enables Saccharomyces cerevisiae and Candida albicans cells to respond to extracellular amino acids. The pathway controls the activity of two homologous transcription factors Stp1 and Stp2 (Stp1/Stp2). In the absence of inducing amino acids, Stp1/Stp2 are maintained latent due to the presence of N-terminal cytoplasmic retention and promoter exclusion motifs. Signaling events initiated by amino acid binding to Ssy1, the plasma membrane-localized receptor component, activates the Ssy5 endoprotease leading to the cleavage of Stp1/Stp2. The cleaved forms of Stp1/Stp2, lacking their N-termini, target the nucleus where they induce SPS-sensor controlled genes.
We addressed unresolved mechanistic questions regarding the transduction of SPS-sensor derived signals in S. cerevisiae. To test spatial parameters that may affect Stp1 cleavage, we fused Stp1 to the C-terminus of the well-characterized ER membrane-localized chaperone Shr3. Expression of the Shr3-Stp1 chimeric protein suppresses both shr3Δ and stp1Δ null phenotypes, indicating that the chimer retains Shr3 chaperone activity and that Ssy5-catalyzed processing of Stp1 occurs even in an artificial membrane-bound context. Subcellular fractionation indicates that the Shr3-Stp1 fusion exclusively localizes to the ER. Significantly, signaling is unabated in a strain that lacks PM-ER junctions in which Ssy1 and the chimer are spatially clearly separated. Thus, once activated by events linked to the plasma membrane, the catalytically competent Ssy5 Cat-domain can relocate to the ER.
We used Candida albicans to assess how amino acid-induced and SPS-sensor-dependent signals are integrated into the central signaling pathways controlling yeast-to-hyphal morphological transitions in this human fungal pathogen. Our results show that the SPS-sensor dependency is indirect, being the consequence of SPS-sensor induced expression of amino acid permease genes, and that the hyphae-inducing effect of amino acids is derived from their metabolism. Specifically, arginine, ornithine and proline are internalized and metabolized to glutamate in the mitochondria, generating NADH and FADH2. These electron carriers are oxidized by mitochondrial respiration generating ATP, which triggers the Ras1/cAMP/PKA pathway to activate the morphogenic effector transcription factor Efg1. Thus, the SPS-sensor is important to maintain the intracellular amino acid pools that ensure proper metabolic control.