Genetic and biochemical characterization of a novel sensor of extracellular amino acids in yeast

Abstract: Eukaryotic cells make developmental decisions that initiate distinct programs of gene expression. In the yeast Saccharomyces cerevisiae the majority of decisions affecting developmental outcomes are made in response to extra- and intracellular derived nutritional signals. The mechanisms by which cells obtain, process and integrate information regarding nutrient levels in the external environment are not fully understood. Cellular utilization of amino acids in yeast provides a system to examine how extracellularly dervied signals affect cellular growth and development. This study describes the cloning and characterization of a novel amino acid sensing system in yeast. A genetic selection for mutations that affect uptake and compartmentalization of amino acids led to the isolation of a unique member of the amino acid permease family, SSY1, and two additional components PTR3 and SSY5. Ssy1p has a unique, extended N-terminal domain that is not present in other amino acid permeases. Ssy5p and Ptr3p are peripherally associated plasma membrane proteins that lack known functional homologues. My results show that in response to amino acids, the Ssylp/Ptr3p/Ssy5p (SPS) sensing system initiates signals that regulate the expression of several amino acid transport and metabolic genes, compartmentalization of amino acids, and filamentous invasive growth. Each of the three components of the SPS-sensor adopt conformations and modifications that are dependent upon the availability of amino acids, and on the presence of the other two components. Rapid physical alterations and reduced levels of sensor components are consistent with their being down-regulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid initiated signals transduced by the SPS sensor. The fact that the components of the SPS sensor lack features of known signal transducing systems implies that this sensor mediates signals by novel mechanisms. Only recently has it become evident that other unique, transporter like proteins (Snf3p, Rgt2p, Mep2p) similarly function to transduce extracellularly derived signals that regulate gene expression. Genome wide transcriptional analysis reveals that SPS mediated amino acid induced signals lead to derepression of a phylogenetically related subset of amino permeases. The lack of response to amino acids in the environment also affects down- stream components of other carbon and nitrogen metabolic pathways, a result of the complex cross-talk between multiple nutritionally derived signals that become integrated in order to regulate cell growth and development. To identify components affecting down-stream targets of the SPS-sensor, spontaneous amino acid sensor independent (asi) suppressor mutations were isolated. Recessive mutations were found in known genes regulating transcription, as well as ubiquitin-dependent endocytic uptake and degradation of amino acid permeases or other aspects of vesicular transport. Mutations in three previously uncharacterized genes, ASI1, ASI2 and ASI3, as well as a dominant mutation in ASI13 efficiently suppressed mutant growth phenotypes and transcriptional defects of SPS-sensor mutations. ASI1 and ASI3 are predicted to encode homologous membrane bound proteins that carry RING-HC domains characteristic of ubiquitin ligases. Further characterization of ASI1/2/3 and ASI13 may unravel novel aspects of gene regulation by ubiquitin mediated processes.