Ssy1 in Saccharomyces cerevisiae is an amino acid receptor evolved from amino acid transporters. It is situated in the plasma membrane in the SPS complex, together with the WD40-repeat protein Ptr3 and the endoprotease Ssy5. Binding of extracellular amino acids to Ssy1 triggers liberation of the catalytic domain of Ssy5, which removes an inhibitory domain from the transcription factor Stp1, freeing it to activate genes encoding amino acid transporters. We mapped 7 constitutively signaling and hyper-responsive SSY1 mutations onto AlphaFold- and Phyre2-based 3D-models of Ssy1 to inform conformational steps involved in signaling. The predictions suggest a model in which an occluded, inward-facing conformation of Ssy1 leads to signaling. The mutations suggest a hinge in TM12 which, combined with a C-terminal 'latch', offers a mechanism for signaling. AlphaFold 3 modeling suggests that conserved sequence boxes in the N-terminal cytoplasmic domain of Ssy1 serve as interaction faces for binding of Ptr3, Ssy5 and casein kinases Yck1 and Yck2 (Yck). In addition, interaction faces between Ptr3 and Ssy5 were predicted. Antagonism between phosphorylation and dephosphorylation of Ptr3 and Ssy5 by Yck and Protein Phosphatase 2A (PP2A) is key in signaling. We found Yck phosphorylation motifs as well as binding motifs for regulatory subunit Rts1 of PP2A, in both Ptr3 and Ssy5. These motifs, together with sites of PTR3 and SSY5 gain-of-function mutations, were mapped onto AlphaFold models of Ptr3 and Ssy5. The results constitute a basis for predicting novel aspects of phosphorylation in the signaling mechanism.

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Journal Article

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Scharff-Poulsen P, Kielland-Brandt MC

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