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Ssy5p is a 77-kDa protein believed to be a component of the SPS amino acid sensor complex in the plasma membrane of Saccharomyces cerevisiae. Ssy5p has been suggested to be a chymotrypsin-like serine protease that activates the transcription factor Stp1p upon exposure of the yeast to extracellular amino acid. Here we overexpressed and partially purified Ssy5p to improve our understanding of its structure and function. Antibodies against Ssy5p expressed in Escherichia coli were isolated and used to detect Ssy5p processing in S. cerevisiae cells. Partial purification and N-terminal sequencing of processed Ssy5p revealed in vivo cleavage of Ssy5p between amino acids 381 and 382. We also isolated constitutively signaling SSY5 mutants and quantified target promoter activation and Stp1p processing. One mutant contained an amino acid substitution in the prodomain, whereas three others harbored amino acid substitutions in the protease domain. Dose-response analysis indicated that all four mutants exhibited increased basal levels of Stp1p processing. Interestingly, whereas the three constitutive mutants mapping to the protease domain of Ssy5p exhibited the decreased 50% effective concentration (EC(50)) characteristic of constitutive mutations previously found in Ssy1p, Ptr3p, and Ssy5p, the EC(50) of the mutation that maps to the prodomain of Ssy5p remained essentially unchanged. In a model of Ssy5p derived from its similarities with alpha-lytic protease from Lysobacter enzymogenes, the sites corresponding to the mutations in the protease domain are clustered in a region facing the prodomain, suggesting that this region interacts with the prodomain and participates in the conformational dynamics of sensing.

eukaryotic cell

Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of <i>Saccharomyces cerevisiae</i> and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics

Mapping of an Internal Protease Cleavage Site in the Ssy5p Component of the Amino Acid Sensor of Saccharomyces cerevisiae and Functional Characterization of the Resulting Pro- and Protease Domains by Gain-of-Function Genetics