Abstract
Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells. In response to extracellular amino acids, Ssy5 proteolytically activates the transcription factors Stp1 and Stp2, leading to enhanced Ssy1-Ptr3-Ssy5 (SPS) sensor-regulated gene expression. Ssy5 comprises a catalytic (Cat) domain and an extensive regulatory prodomain. Ssy5 is refractory to both broad-spectrum and serine protease-specific inhibitors, confounding its classification as a protease, and no information about Ssy5's cleavage-site preferences and its mechanism of substrate selection is available. Here, using mutational and inhibition experiments, we investigated the biogenesis and catalytic properties of Ssy5 and conclusively show that it is a serine protease. Atypical for the majority of serine proteases, Ssy5's prodomain was obligatorily required in cis during biogenesis for the maturation of the proteolytic activity of the Cat domain. Autolysis and Stp1 and Stp2 cleavage occurred between a cysteine (at the P1 site) and a serine or alanine (at the P'1 site) and required residues with short side chains at the P1 site. Substitutions in the Cat domain affecting substrate specificity revealed that residues Phe-634, His-661, and Gly-671 in the S1-binding pocket of this domain are important for Ssy5 catalytic function. This study confirms that the signaling protease Ssy5 is a serine protease and provides a detailed understanding of the biogenesis and intrinsic properties of this key enzyme in yeast.
Highlights
Ssy5 is a signaling endoprotease that plays a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival of yeast (Saccharomyces cerevisiae) cells
We report under sensitive assay conditions that the catalytic activity of Ssy5 is responsive to the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF), a finding that conclusively confirms that Ssy5 is a serine protease
When grown on yeast extract/peptone/dextrose (YPD) media, cells lacking a functional Ssy5 are resistant to AzC and sensitive to MM; mutant cells do not take up the toxic proline analog and are unable to take up sufficient branched-chain amino acids to support growth
Summary
Global Business Development and Licensing Consumer Healthcare, Sanofi-Aventis Deutschland GmbH, 65926 Frankfurt am Main, Germany. The prodomain remains associated with the Cat domain and prevents substrate processing [16, 17]. The prodomain functions as the inhibitory subunit of the SPS sensor At this stage Ssy constitutes a primed protease able to bind but not proteolytically process Stp and Stp, the only known substrates. Similar to Ssy5, ␣-lytic protease is expressed with an N-terminal prodomain essential for the catalytic domain to reach a correctly folded and active state (18 –20). The ␣-lytic protease prodomain remains strongly associated with the catalytic domain, sterically blocking the active site until secreted, at which point the inhibitory prodomain is degraded by extracellular proteases [21]. The data provide a detailed understanding of the intrinsic sequence requirements within the catalytic site that are important for autolysis during biogenesis and processing of Stp and Stp. We have identified the scissile bonds in Stp and Stp and established the rules for their recognition as substrates, and we analyzed the residues in the substrate-binding pocket involved in substrate recognition
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