Ligand-activated proteolysis in nutrient signaling

Abstract

Cells respond to changing nutrient availability and make adjustments in physiological processes. Central for making proper adjustments is the ability to execute appropriate changes in patterns of gene expression.

The budding yeast Saccharomyces cerevisiae, responds to the presence of extracellular amino acids by up regulating systems that internalize these nutrients. Extracellular amino acids are sensed by the amino acid transporter-like receptor Ssy1p that is localized to the plasma membrane. Ssy1p generates a signal that is transmitted via a pathway minimally composed of the core components Ssy1p, Ptr3p, and Ssy5p (SPS). The SPS sensor pathway ultimately up regulates genes encoding amino acid transporters, also known as amino acid permeases.

This thesis describes the elucidation of the mechanism connecting amino acid induced signals generated at the plasma membrane and gene regulation in the nucleus. A genetic selection for mutations that enable amino acid permease genes to be expressed even in the absence of a functional SPS sensor pathway identified the dominant ASI13-1 mutation. The ASI13-1 gene encodes a constitutively active form of the transcription factor Stp1p that lacks its regulatory N-terminal domain. Stp1p and its close homologue Stp2p are synthesized as latent cytoplasmic precursors. In response to extracellular amino acids, the SPS sensor induces the rapid endoproteolytic processing of Stp1p and Stp2p. The shorter forms of these transcription factors, lacking N-terminal inhibitory domains, are targeted to the nucleus, where they transactivate SPS-sensor target genes.

Several genetic approaches have been applied to identify mutations that affect the SPS sensor pathway. A novel genetic selection specifically designed for rare mutations that affect the SPS-sensing pathway identified the F-box protein Grr1p as an obligatory factor required for Stp1p and Stp2p processing. Genetic analysis suggests that Grr1p has a role in signal transduction within the SPS sensor.

The N-terminal domains of Stp1p and Stp2p contain two conserved motifs that are required for proper nuclear exclusion and proteolytic processing. These motifs function in parallel; mutations that abolish processing inhibit signaling, whereas mutations that interfere with cytoplasmic retention result in constitutive activation of SPS sensorregulated genes independently of processing. The N-terminal domain of Stp1p is functionally autonomous and transferable to other transcription factors, where its presence confers regulated nuclear exclusion and SPS sensor-induced proteolytic processing.

Proteolytic processing of recombinant Stp1p in cell free lysates supports the notion of a SPS sensor activated protease. Analysis indicates that Ssy5p is a chymotrypsin-like serine protease that is activated via the SPS sensor pathway and is responsible for Stp1p and Stp2p processing. Mutations in the predicted catalytic triad of Ssy5p abolish Stp1p processing. A constitutive SSY5 mutant promotes processing of Stp1p even in the absence of amino acids or Ssy1p and Ptr3p. Finally, Stp1p is processed when heterologously coexpressed together with activated Ssy5p in Schizosaccharomyces pombe, an organism that lacks the SPS sensor pathway.

Taken together, these results define a unique and streamline metabolic control pathway that directly routes nutrient signals initiated at the plasma membrane to transcriptional activation in the nucleus.