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Regulatory mechanisms of amino acid-induced signaling in Saccharomyces cerevisiae
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes studies aimed at elucidating the molecular mechanisms that regulate the SPS (Ssy1-Ptr3-Ssy5) signal transduction pathway in the yeast Saccharomyces cerevisiae. This pathway is induced by extracellular amino acids and facilitates their uptake. The most downstream effectors of the SPS pathway, the homologous transcription factors Stp1 and Stp2 (Stp1/2), are synthesized as latent precursors with N-terminal regulatory domains that restrict their nuclear accumulation. Amino acid-induced signaling, initiated by the plasma membrane localized receptor Ssy1, leads via Ptr3 to the activation of the endoprotease Ssy5. Active Ssy5 cleaves the regulatory domains in Stp1/2. As a consequence, the processed transcription factors lacking their N-terminal domains accumulate in the nucleus and activate the transcription of amino acid permease genes to enhance the uptake capacity of cells.

Ssy5 is synthesized as a zymogen precursor that processes itself into a prodomain and catalytic (Cat) domain that remain non-covalently associated. We found that the prodomain functions as an inhibitor of the Cat domain. Signaling triggers the degradation of the prodomain by the proteasome, thereby releasing Cat domain activity (paper I). We identified a motif in the prodomain that functions as inducible phosphodegron. Upon signaling, this motif is phosphorylated which triggers prodomain polyubiquitylation, and as a consequence, its proteasomal degradation (paper II). Also, we found that Ptr3 functions to mediate prodomain phosphorylation upon signaling and that protein phosphatase 2A constitutively mutes phosphorylation-dependent activation of Ssy5 (paper III).

Finally, in addition to the regulation of the processing protease Ssy5, the control of transcriptional activity of Stp1 depends on a motif within its N-terminal regulatory domain, designated Region I. We found that Region I mediates latency by functioning as cytoplasmic retention determinant and nuclear degron (paper IV).

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2014. , 70 p.
Keyword [en]
Signal transduction, regulatory mechanisms, nutrient sensing, Saccharomyces cerevisiae
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-97329ISBN: 978-91-7447-830-3 (print)OAI: oai:DiVA.org:su-97329DiVA: diva2:676976
Public defence
2014-02-04, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-01-13 Created: 2013-12-08 Last updated: 2014-01-02Bibliographically approved
List of papers
1. The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp1
Open this publication in new window or tab >>The prodomain of Ssy5 protease controls receptor-activated proteolysis of transcription factor Stp1
Show others...
2010 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 30, no 13, 3299-309 p.Article in journal (Refereed) Published
Abstract [en]

Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells

National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-44307 (URN)10.1128/MCB.00323-10 (DOI)000278626100013 ()20421414 (PubMedID)
Available from: 2010-11-05 Created: 2010-11-05 Last updated: 2017-12-12Bibliographically approved
2. A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5
Open this publication in new window or tab >>A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5
2011 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 22, no 15, 2754-2765 p.Article in journal (Refereed) Published
Abstract [en]

Regulated proteolysis serves as a mechanism to control cellular processes. The SPS (Ssy1-Ptr3-Ssy5) sensor in yeast responds to extracellular amino acids by endoproteolytically activating transcription factors Stp1 and Stp2 (Stp1/2). The processing endoprotease Ssy5 is regulated via proteasomal degradation of its noncovalently associated N-terminal prodomain. We find that degradation of the prodomain requires a conserved phosphodegron comprising phosphoacceptor sites and ubiquitin-accepting lysine residues. Upon amino acid induction, the phosphodegron is modified in a series of linked events by a set of general regulatory factors involved in diverse signaling pathways. First, an amino acid-induced conformational change triggers phosphodegron phosphorylation by the constitutively active plasma membrane-localized casein kinase I (Yck1/2). Next the prodomain becomes a substrate for polyubiquitylation by the Skp1/Cullin/Grr1 E3 ubiquitin ligase complex (SCF(Grr1)). Finally, the modified prodomain is concomitantly degraded by the 26S proteasome. These integrated events are requisite for unfettering the Ssy5 endoprotease, and thus Stp1/2 processing. The Ssy5 phosphoacceptor motif resembles the Yck1/2- and Grr1-dependent degrons of regulators in the Snf3/Rgt2 glucose-sensing pathway. Our work defines a novel proteolytic activation cascade that regulates an intracellular signaling protease and illustrates how general signaling components are recruited to distinct pathways that achieve conditional and specific signaling outputs.

National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-65625 (URN)10.1091/mbc.E11-04-0282 (DOI)000293227200009 ()21653827 (PubMedID)
Note

authorCount :4

Available from: 2011-12-13 Created: 2011-12-13 Last updated: 2017-12-08Bibliographically approved
3. Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I
Open this publication in new window or tab >>Rts1-protein phosphatase 2A antagonizes Ptr3-mediated activation of the signaling protease Ssy5 by casein kinase I
2013 (English)In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 24, no 9, 1480-1492 p.Article in journal (Refereed) Published
Abstract [en]

Ligand-induced conformational changes of plasma membrane receptors initiate signals that enable cells to respond to discrete extracellular cues. In response to extracellular amino acids, the yeast Ssy1-Ptr3-Ssy5 sensor triggers the endoproteolytic processing of transcription factors Stp1 and Stp2 to induce amino acid uptake. Activation of the processing protease Ssy5 depends on the signal-induced phosphorylation of its prodomain by casein kinase I (Yck1/2). Phosphorylation is required for subsequent Skp1/Cullin/Grr1 E3 ubiquitin ligase-dependent polyubiquitylation and proteasomal degradation of the inhibitory prodomain. Here we show that Rts1, a regulatory subunit of the general protein phosphatase 2A, and Ptr3 have opposing roles in controlling Ssy5 prodomain phosphorylation. Rts1 constitutively directs protein phosphatase 2A activity toward the prodomain, effectively setting a signaling threshold required to mute Ssy5 activation in the absence of amino acid induction. Ptr3 functions as an adaptor that transduces conformational signals initiated by the Ssy1 receptor to dynamically induce prodomain phosphorylation by mediating the proximity of the Ssy5 prodomain and Yck1/2. Our results demonstrate how pathway-specific and general signaling components function synergistically to convert an extracellular stimulus into a highly specific, tuned, and switch-like transcriptional response that is critical for cells to adapt to changes in nutrient availability.

National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-92273 (URN)10.1091/mbc.E13-01-0019 (DOI)000321132100020 ()
Funder
Swedish Research Council
Note

AuthorCount:2;

Available from: 2013-07-25 Created: 2013-07-25 Last updated: 2017-12-06Bibliographically approved
4. Latency of Transcription Factor Stp1 Depends on a Modular Regulatory Motif that Functions as Cytoplamsic Retention Determinant and Nuclear Degron
Open this publication in new window or tab >>Latency of Transcription Factor Stp1 Depends on a Modular Regulatory Motif that Functions as Cytoplamsic Retention Determinant and Nuclear Degron
(English)Manuscript (preprint) (Other academic)
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-97324 (URN)
Available from: 2013-12-08 Created: 2013-12-08 Last updated: 2013-12-09

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