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In vivo analysis of amino acid permease folding in yeast
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2019. , p. 54
Keywords [en]
Nutrient sensing, signal transduction, endoprotease, subcellular fractionation, membrane-localized chaperone, amino acid permease, endoplasmic reticulum, membrane protein folding, Saccharomyces cerevisiae
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-171561ISBN: 978-91-7797-771-1 (print)ISBN: 978-91-7797-772-8 (electronic)OAI: oai:DiVA.org:su-171561DiVA, id: diva2:1342924
Public defence
2019-09-27, sal E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 3: Submitted.

Available from: 2019-09-04 Created: 2019-08-14 Last updated: 2019-08-28Bibliographically approved
List of papers
1. The ER membrane chaperone Shr3 co-translationally assists biogenesis of related polytopic membrane protein substrates
Open this publication in new window or tab >>The ER membrane chaperone Shr3 co-translationally assists biogenesis of related polytopic membrane protein substrates
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Polytopic membrane proteins with multiple transmembrane segments (TMS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Discrete sets of polytopic membrane proteins in Saccharomyces cerevisiae require ER membrane-localized chaperones (MLC) to prevent aggregation and fold properly. Shr3, the best characterized MLC, is specifically required for the functional expression of amino acid permeases (AAP), a family of transport proteins comprised of twelve TMS. We performed comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with split-ubiquitin approaches to probe chaperone-substrate (Shr3-AAP) interactions in vivo. We report a surprisingly low level of sequence specificity underlies Shr3-AAP interactions, which initiate after the first 2 to 4 TMS of AAP partition into the membrane. The Shr3-AAP interactions successively strengthen and then weaken as all 12 TMS are inserted. Thus, Shr3 acts transiently in a co-translationally manner to prevent TMS of translation intermediates from engaging in non-productive interactions, thereby preventing AAP misfolding during biogenesis.

National Category
Biological Sciences
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-171558 (URN)
Available from: 2019-08-14 Created: 2019-08-14 Last updated: 2019-08-19Bibliographically approved
2. Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of PM‐ER junctions
Open this publication in new window or tab >>Ssy1 functions at the plasma membrane as a receptor of extracellular amino acids independent of PM‐ER junctions
2019 (English)In: Traffic: the International Journal of Intracellular Transport, ISSN 1398-9219, E-ISSN 1600-0854Article in journal (Refereed) Epub ahead of print
Abstract [en]

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.

Keywords
Subcellular fractionation, nutrient sensing, amino acid permease, signal transduction, ER-PM tethering, Shr3, membrane-localized chaperone, Saccharomyces cerevisiae
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-160466 (URN)10.1111/tra.12681 (DOI)
Available from: 2018-09-25 Created: 2018-09-25 Last updated: 2019-08-14
3. Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling protease
Open this publication in new window or tab >>Spatial and temporal regulation of the endoproteolytic activity of the SPS-sensor controlled Ssy5 signaling protease
Show others...
(English)In: Article in journal (Refereed) Submitted
Abstract [en]

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.

Keywords
Nutrient sensing, signal transduction, regulated proteolysis, endoprotease, Saccharomyces cerevisiae
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-160478 (URN)
Available from: 2018-09-25 Created: 2018-09-25 Last updated: 2019-08-14Bibliographically approved

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