12345671 of 15
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Polytopic membrane protein biogenesis from the perspective of a specialized membrane-localized chaperone
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.ORCID iD: 0009-0002-3935-7886
2025 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In eukaryotic cells the early steps of polytopic membrane protein biogenesis take place at the endoplasmic reticulum (ER). Complex polytopic membrane proteins with multiple membrane-spanning segments (MS) co-translationally insert into the ER membrane and fold into native states prior to being recognized as cargo for incorporation, or packaging, into COPII-coated ER-derived secretory vesicles. Successful progression through these early steps is a requisite for the ultimate delivery and functional expression of membrane proteins at appropriate cellular membranes. The list of biogenesis factors that contribute to membrane protein biogenesis is expanding, and due to their diversity, it is imperative to investigate their individual functions. Highly specialized membrane-localized chaperones (MLC) have been identified that are required for the functional expression of discrete sets of related membrane protein substrates. This thesis focuses on Shr3 in Saccharomyces cerevisiae. Shr3 was the first MLC defined and was identified based on it being required for the biogenesis of all eighteen members of the Amino Acid Permease (AAP) protein family, each comprised of 12 membrane-spanning (MS) segments. Despite numerous independent findings corroborating the co-translational nature of the Shr3-AAP interactions during AAP translation, ER membrane insertion and folding, direct formal proof has been elusive. Specifically, a detailed mechanistic understanding of how Shr3 facilitates AAP folding has been lacking, and the underlying mechanisms governing its strict ER membrane localization have not been rigorously investigated. The studies documented in this thesis were aimed to obtain a deeper mechanistic understanding of how Shr3 facilitates AAP folding (Paper I) and its strict ER localization (Paper II) and to directly probe the co-translational interaction of Shr3 with its AAP folding substrates (Paper III).

Specifically, in Paper I, the chaperone folding function and temporal requirement of Shr3 was investigated. Strikingly, the experiments revealed that relatively few amino acids within the 4 MS segments of Shr3 contribute to substrate specific interactions, however, mutations within the 2 luminal loops of Shr3 did manifest substrate specific effects. Further, a split-ubiquitin approach was used to probe interactions between Shr3 and MS segments of AAP. The data indicate that Shr3 interacts early with the first few MS of AAP, presumably directly as they partition into the ER membrane. The experiments documented in Paper II were directed at identifying ER retention determinants present in the cytoplasmic carboxy-terminal tail of Shr3. Retention was found to depend on multiple motifs that function in an additive fashion. Surprisingly, one of the motifs was close to the membrane domain. This unexpected finding provided a novel tool that was exploited to obtain the first evidence of a mechanistic coupling between Shr3-dependent folding and packaging of AAP into COPII-coated vesicles. Paper III describes the use of a translational arrest peptide capable of stalling AAP translation on the ribosome. Strikingly, Shr3 was found associated with a nascent AAP chain with 10 membrane-spanning segments. This finding provides formal proof that Shr3 indeed functions in a co-translational manner. Collectively, these studies offer a significantly enhanced mechanistic understanding of MLC and their requirement during the biogenesis of complex polytopic membrane proteins

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2025. , p. 65
Keywords [en]
polytopic membrane protein, selective membrane-localized chaperone, co-translational interaction, folding, packaging, endoplasmic reticulum, split-ubiquitin, arrest peptide, amino acid permease, transport, Shr3, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-237522ISBN: 978-91-8107-066-8 (print)ISBN: 978-91-8107-067-5 (electronic)OAI: oai:DiVA.org:su-237522DiVA, id: diva2:1924532
Public defence
2025-02-20, Vivi Täckholmsalen (Q-salen) NPQ-huset, Svante Arrhenius väg 20, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2025-01-28 Created: 2025-01-06 Last updated: 2025-01-21Bibliographically approved
List of papers
1. ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis
Open this publication in new window or tab >>ER-localized Shr3 is a selective co-translational folding chaperone necessary for amino acid permease biogenesis
2023 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 222, no 9, article id e202208060Article in journal (Refereed) Published
Abstract [en]

Proteins with multiple membrane-spanning segments (MS) co-translationally insert into the endoplasmic reticulum (ER) membrane of eukaryotic cells. Shr3, an ER membrane–localized chaperone in Saccharomyces cerevisiae, is required for the functional expression of a family of 18 amino acid permeases (AAP) comprised of 12 MS. We have used comprehensive scanning mutagenesis and deletion analysis of Shr3 combined with a modified split-ubiquitin approach to probe chaperone–substrate interactions in vivo. Shr3 selectively interacts with nested C-terminal AAP truncations in marked contrast to similar truncations of non-Shr3 substrate sugar transporters. Shr3–AAP interactions initiate with the first four MS of AAP and successively strengthen but weaken abruptly when all 12 MS are present. Shr3–AAP interactions are based on structural rather than sequence-specific interactions involving membrane and luminal domains of Shr3. The data align with Shr3 engaging nascent N-terminal chains of AAP, functioning as a scaffold to facilitate folding as translation completes.

National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:su:diva-227688 (URN)10.1083/jcb.202208060 (DOI)001177877100001 ()37477900 (PubMedID)2-s2.0-85165774675 (Scopus ID)
Available from: 2024-04-05 Created: 2024-04-05 Last updated: 2025-01-06Bibliographically approved
2. The membrane chaperone Shr3 couples amino acid permease folding and ER exit
Open this publication in new window or tab >>The membrane chaperone Shr3 couples amino acid permease folding and ER exit
(English)Manuscript (preprint) (Other academic)
National Category
Cell Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-237521 (URN)
Available from: 2025-01-06 Created: 2025-01-06 Last updated: 2025-01-06
3. Development of a translational arrest peptide-based system to probe co-translational interactions of an ER-integral chaperone in yeast
Open this publication in new window or tab >>Development of a translational arrest peptide-based system to probe co-translational interactions of an ER-integral chaperone in yeast
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:su:diva-237520 (URN)
Available from: 2025-01-06 Created: 2025-01-06 Last updated: 2025-01-06

Open Access in DiVA

Polytopic membrane protein biogenesis from the perspective of a specialized membrane-localized chaperone(7530 kB)39 downloads
File information
File name FULLTEXT01.pdfFile size 7530 kBChecksum SHA-512
4370a13a16cd561c130d21c10e7d7622f7a6c365cabf1505b705fe57353e8648372a6601991e3f3525ad5fc6193eb6b97c4b36f1dd8487229d93f625ae241d5f
Type fulltextMimetype application/pdf

Authority records

Myronidi, Ioanna

Search in DiVA

By author/editor
Myronidi, Ioanna
By organisation
Department of Molecular Biosciences, The Wenner-Gren Institute
Biochemistry and Molecular BiologyCell Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 39 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 367 hits
12345671 of 15
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf