Polytopic membrane protein biogenesis from the perspective of a specialized membrane-localized chaperone
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
2025-01-282025-01-062025-01-21Bibliographically approved
List of papers