Cells continuously sense and respond to changes in the presence, quality and quantity of external and internal nutrients. Specific signaling proteases have been identified based on their roles in processing or destruction of distinct sets of downstream effector proteins in response to environmental cues. The Saccharomyces cerevisiae Ssy5 signaling endoprotease has a key role in regulating central metabolism, cellular aging, and morphological transitions important for growth and survival. Ssy5 is a core component of the Ssy1–Ptr3-Ssy5 (SPS) sensor, which enables yeast cells to respond to extracellular amino acids and induce their uptake. Ssy5 cleaves transcription factors Stp1 and Stp2, permitting their translocation to the nucleus where they enhance the expression of amino acid permease genes. This thesis focuses on Ssy5, its biogenesis and catalytic properties (paper I), the spatial determinants underlying Ssy5 function in SPS-sensor context (paper II) and substrate cleavage (paper III).

Ssy5 is comprised of pro- and catalytic-(Cat)-domains. The Cat-domain possesses characteristic hallmarks of a serine protease; however, serine protease-specific inhibitors have limited effect, confounding its classification. In paper I we unambiguously show that Ssy5 is a serine protease, define the precise sites of cleavage in Stp1 and Stp2, and describe the sequence specific requirements of their cleavage. The uniquely large prodomain (381 amino acids) has two essential functions. Initially, it is required in cis for the maturation of the Cat-domain, helping to overcome a folding barrier that is reflected in the high stability of the Cat-domain. Subsequent to attaining enzymatic competence, Ssy5 undergoes an autolytic cleavage event. The domains remain associated and the prodomain functions to fetter the proteolytic activity of the Cat-domain.

The plasma membrane (PM) localization of Ssy1 has recently been questioned in a report that postulated that Ssy1 is a component of the endoplasmic reticulum (ER) and contributes to the formation of ER-PM junctions. In paper II, using mutational and subcellular fractionation experiments we critically examined this notion that is inconsistent with the current understanding of Ssy5 activation, i.e., the unfettering of the Cat-domain occurs in strict association with Ssy1 at the PM. The data show that Ssy1 is indeed a PM protein, and importantly, Ssy5-activation occurs independent of ER-PM junctions. A di-acidic ER exit motif was identified that is critical for proper PM localization and function of Ssy1. In paper III, we report that the Cat-domain is post-translationally modified in a manner dependent on Ptr3 and the PM casein kinase I (Yck1/2), consistent with Ssy5 activation occurring at the PM. Strikingly, the activated Cat-domain is capable of properly cleaving Stp1 fused to an ER membrane protein. The amino acid-induced cleavage of this artificial membrane-bound substrate occurs in a Δtether strain (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ) lacking ER-PM junctions. These findings indicate that the activated Cat-domain can bind and functionally interact with substrates distant from the PM. Finally, we show that the Cat-domain is degraded faster in amino acid-induced cells. These findings provide novel insights into the SPS-sensing pathway and demonstrate for the first time that the resetting of the SPS-sensing system correlates with Cat-domain degradation.

Plasma membrane (PM) proteins are critical for cells to respond to environmental cues, such as the availability of nutrients. The yeast Saccharomyces cerevisiae is able to sense extracellular amino acids using the SPS sensing system. Activation of the multimeric PM-localized SPS(Ssy1-Ptr3-Ssy5)-sensor complex occurs upon binding of external amino acids to Ssy1, inducing a conformational change. In a Ptr3-mediated event, the catalytic activity of the Ssy5 endoprotease is unfettered, leading to the proteolytic processing of two latent transcription factors, Stp1 and Stp2. Ssy1, the primary sensor component, is a non-transporting member of the amino acid permease (AAP) family of transport proteins, a family of eighteen complex integral membrane proteins comprised of 12 transmembrane segments (TMS). The AAPs exhibit a common requirement for the endoplasmic reticulum (ER)-localized membrane chaperone Shr3 to fold and to be transported to the PM. The absence of Shr3 leads to the accumulation of misfolded AAP species that are targeted for ER-associated degradation. Thus, proper Shr3 function is required as the most upstream and most downstream component of the SPS sensing system. In paper I, we investigate the chaperone function of Shr3. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions. We used a split-ubiquitin approach to probe Shr3-AAP interactions in vivo. The Shr3-AAP interactions initiate early after the first two-to-four TMS of AAPs insert into the ER membrane, successively strengthening and then diminishing after all 12 TMS partition into the membrane. In paper II, we clarified the localization and trafficking determinants of Ssy1. A study by Kralt et al. 2015 reported that Ssy1 primarily localizes to the ER and is sorted to ER-PM tethers. These reported findings are clearly incompatible with the accepted model of amino acid sensing by the SPS-sensor. We critically re-examined the localization of Ssy1 and found that it indeed localizes to the PM, and importantly does so independent of ER-PM tethers. We also identified a novel ER exit motif in the carboxy-terminal tail of Ssy1 required for proper PM localization and SPS-sensor function. In paper III, we report that Ssy5 is able to cleave substrates in unusual contexts, i.e., an engineered substrate carrying rearranged recognition and cleavage determinants placed ectopically at the carboxy terminus of Stp1, and an ER-anchored substrate with Stp1 fused to the carboxy terminus of Shr3. Strikingly, Ssy5 catalyzed cleavage of Shr3-Stp1 in cells lacking ER-PM tethers, indicating that once activated, Ssy5 can find and cleave substrates distant from the PM. Consequently, cells must be able to rein in the activity of the Ssy5 protease to prevent spurious and improper proteolysis. Consistent with this notion, we report that the catalytic domain of Ssy5 is ubiquitylated in a Ptr3 and Yck1/2 dependent manner, and under amino acid-inducing conditions is subject to degradation. We propose a model that degradation of the Ssy5 catalytic domain is essential for resetting the SPS sensing system and a requisite for cells to regain the ability to correctly sense extracellular amino acids.