Amino acid signalling in yeast: functional analysis of the Stp transcription factors

The main subject of this thesis’s work is the external amino acid sensing system in S. cerevisiae. The detection of extracellular amino acids in yeast begins with a transporter homologue devoid of any uptake activity, the Ssy1 sensor. The binding of extracellular amino acids to Ssy1 leads to the successive activation of Ptr3 and the Ssy5 endoprotease. This endoporotease catalyses the processing of two transcriptions factors: Stp1 and Stp2. The Stp factors, released from their N-terminal cytoplasmic-anchored domains, are then translocated into the nucleus, where they activate the transcription of several amino acid permease genes (e.g. AGP1 and DIP5). Starting this work, the Stp factors were considered as functionally redundant.

We first determined that the STP1 and STP2 genes derivate from the event of WGD. The conservation of these two genes in S. cerevisiae was accompanied by a functional divergence of their products at several levels: processing sensibility, transcriptional activation capacity, target genes, cellular abundance level and stability. The Stp2 factor with its high abundance in the cells and its higher Ssy5-processing sensibility is specialized towards induction of the AGP1 gene when the external amino acid signaling is weakly stimulated. Under strong stimulation conditions, the amino acids induce cleavage-triggered destabilization of Stp2 through the proteasomal pathway and the induction of AGP1 is mediated mainly by the Stp1 transcription factor. Unlike Stp2, the Stp1 factor is characterized by its high transcriptional activation capacity and weaker sensitivity towards Ssy5-processing. The Stp factors differ also by their genetic targets. Indeed, only Stp2 regulates the expression of DIP5. Finally, we determined that the processing sensibility and the transcriptional activation capacity of each Stp factors is directly linked to their N- and C-terminal domains, respectively.

The phosphorylation states and the degradation of the Stp2 factor were also examined. The event of degradation concerns only the processed forms of this factor and takes places principally in the nucleus. Some data indicate that such an event might be important to limit the activation capacity of this factor. The role of the Stp2 phosphorylation in the external amino acid signaling pathway is still unknown but this event might be important for the Stp2 degradation or its transcriptional activity.

The unique Stp factor from Kluyveromyces lactis (Kl-Stp), a pre-WGD species, was also studied. The Kl-Stp factor shares at least two characteristic with the S. cerevisiae Stp2 factor: high sensibility towards processing and high levels of degradation. This observation leads us to conclude to that the STP genes may have been conserved after WGD though a mechanism called neofunctionalization (one of the duplicate obtained after duplication retains the ancestral function while the other evolves to perform a novel function).

Finally, a new model for the external amino acid signaling pathway that brings together all the data obtained during this thesis’s work is proposed.