# Structural bases of signal generation and transduction by the SPS amino acid sensor of Saccharomyces cerevisiae

## TL;DR

This paper explores how a yeast amino acid sensor works by using structural models to understand its signaling mechanism.

## Contribution

The study provides a structural model of the SPS complex and identifies key conformational and phosphorylation events in signaling.

## Key findings

- Constitutively signaling SSY1 mutations suggest a hinge in TM12 and a C-terminal latch for signaling.
- AlphaFold models reveal interaction faces for Ssy1 with Ptr3, Ssy5, and casein kinases Yck1/Yck2.
- Phosphorylation and dephosphorylation of Ptr3 and Ssy5 by Yck and PP2A are critical for signaling.

## Abstract

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.

Graphical AbstractFor image description, please refer to the figure legend and surrounding text.