| 1. |
- Martins, António, 1976-, et al.
(author)
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Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling protease
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Journal article (peer-reviewed)abstract
- The Saccharomyces cerevisiae Ssy5 signaling protease is a core component of the plasma membrane (PM)-localized SPS (Ssy1-Ptr3-Ssy5)-sensor. In response to extracellular amino acids, the SPS-sensor orchestrates the proteasomal degradation of the inhibitory Ssy5 prodomain. The unfettered catalytic (Cat)-domain cleaves latent transcription factors Stp1 and Stp2, freeing them from negative N-terminal regulatory domains. By studying the spatial and temporal constraints affecting the unfettered Cat-domain, we found that it can cleave substrates not associated with the PM; the Cat-domain efficiently cleaves Stp1 even when fused to the carboxy-terminal of the endoplasmic reticulum (ER) membrane protein Shr3. The amino acid-induced cleavage of this synthetic membrane-anchored substrate occurs in a Δtether strain lacking ER-PM junctions. We report that the bulk of the Cat-domain is soluble, exhibits a disperse intracellular distribution and is subject to ubiquitylation. Cat-domain ubiquitylation is dependent on Ptr3 and the integral PM casein kinase I (Yck1/2). Time-course experiments reveal that the non- and ubiquitylated forms of the Cat-domain are stable in cells grown in the absence of inducing amino acids. By contrast, amino acid induction significantly accelerates Cat-domain degradation. These findings provide novel insights into the SPS-sensing pathway and suggest that Cat-domain degradation is a requisite for resetting SPS-sensor signaling.
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| 2. |
- Martins, António, 1976-
(author)
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The cell biology and catalytic properties of the nutrient-induced signaling endoprotease Ssy5
- 2018
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Doctoral thesis (other academic/artistic)abstract
- 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.
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| 3. |
- Ring, Andreas, et al.
(author)
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Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of plasma membrane‐endoplasmic reticulum junctions
- 2019
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In: Traffic. - : Wiley. - 1398-9219 .- 1600-0854. ; 20:10, s. 775-784
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Journal article (peer-reviewed)abstract
- Evidence from multiple laboratories have implicated Ssy1, a non‐transporting amino acid permease, as the receptor component of the yeast plasma membrane (PM)‐localized SPS (Ssy1‐Ptr3‐Ssy5)‐sensor. Upon binding external amino acids, Ssy1 is thought to initiate signaling events leading to the induction of amino acid permease gene expression. In striking contrast, Kralt et al. 2015 (Traffic 16:135‐147) have questioned the role of Ssy1 in amino acid sensing and reported that Ssy1 is a component of the endoplasmic reticulum (ER), where it reportedly participates in the formation of ER‐PM junctions. Here, we have re‐examined the intracellular location of Ssy1 and tested the role of ER‐PM junctions in SPS sensor signaling. We show that the C‐terminal of Ssy1 carries a functional ER‐exit motif required for proper localization of Ssy1 to the PM. Furthermore, ER‐PM junctions are dispensable for PM‐localization and function of Ssy1; Ssy1 localizes to the PM in a Δtether strain lacking ER‐PM junctions (ist2Δ scs2Δ scs22Δ tcb1Δ tcb2Δ tcb3Δ), and this strain retains the ability to initiate signals induced by extracellular amino acids. The data demonstrate that Ssy1 functions as the primary amino acid receptor and that it carries out this function at the PM.
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