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Ssy5 is a signaling serine protease that exhibits atypical biogenesis and marked S1 specificity
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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Number of Authors: 72018 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, no 22, p. 8362-8378Article in journal (Refereed) Published
Abstract [en]

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 P1 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.

Place, publisher, year, edition, pages
2018. Vol. 293, no 22, p. 8362-8378
Keywords [en]
Saccharomyces cerevisiae, serine protease, signal transduction, substrate specificity, yeast, enzyme structure, environmental sensing, receptor activated proteolysis, signaling protease, SPS sensor, zymogen, signaling protease
National Category
Biological Sciences
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-157683DOI: 10.1074/jbc.RA118.002457ISI: 000434205700004PubMedID: 29661936OAI: oai:DiVA.org:su-157683DiVA, id: diva2:1236028
Available from: 2018-07-30 Created: 2018-07-30 Last updated: 2018-09-27Bibliographically approved
In thesis
1. The cell biology and catalytic properties of the nutrient-induced signaling endoprotease Ssy5
Open this publication in new window or tab >>The cell biology and catalytic properties of the nutrient-induced signaling endoprotease Ssy5
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2018. p. 55
Keywords
Nutrient sensing, signal transduction, regulated proteolysis, endoprotease, serine protease, substrate specificity, subcellular fractionation, Saccharomyces cerevisiae
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-160485 (URN)978-91-7797-432-1 (ISBN)978-91-7797-433-8 (ISBN)
Public defence
2018-11-13, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Available from: 2018-10-19 Created: 2018-09-27 Last updated: 2018-10-16Bibliographically approved

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Martins, AntónioPfirrmann, ThorstenAndréasson, ClaesLjungdahl, Per O.
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