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Activation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-2930-801X
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In S. cerevisiae the transmembrane protein Respiratory Supercomplex Factor 1 (Rcf1) is involved in formation of the cytochrome c oxidase - bc1 supercomplex. It has also been suggested to mediate electron transfer between the two respiratory enzymes via interactions with cytochrome c. Removal of Rcf1 results in decreased CytcO activity as well as a decrease in the fraction of supercomplexes. The Rcf1 protein can presumably be found as both a monomer and dimer in the membrane. A structure of the latter has been determined using NMR. In this study, we show that co-reconstitution of purified Rcf1 with CytcO from a rcf1Δ strain in liposomes yielded an increase in the CytcO activity. Also, reconstitution of Rcf1 in sub-mitochondrial particles from the rcf1Δ strain yielded an increase in the CytcO activity. However, the increased activity was only observed when the Rcf1 protein was fully unfolded and then refolded in the presence of a membrane. Collectively, the data indicate that Rcf1 can be reconstituted in a membrane as a dimer, but the protein can interact with and reactivate CytcO only in the monomeric form.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-173584OAI: oai:DiVA.org:su-173584DiVA, id: diva2:1354796
Available from: 2019-09-26 Created: 2019-09-26 Last updated: 2019-09-26Bibliographically approved
In thesis
1. Protein and lipid interactions within the respiratory chain: Studies using membrane-mimetic systems
Open this publication in new window or tab >>Protein and lipid interactions within the respiratory chain: Studies using membrane-mimetic systems
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Energy conversion from nutrients to ATP is a vital process in cells. The process, called oxidative phosphorylation (OXPHOS) is performed by a combination of membrane-bound proteins. These proteins have been studied in great detail in the past, however much is still unknown about how they interact with each other. Studying the OXPHOS proteins in their native environment can be difficult due to the complexity of living cells. By isolating parts of the OXPHOS system and inserting them into membrane-mimetic systems it is possible to investigate their functions in a controlled environment.

In the work presented here, we co-reconstituted several of these proteins into liposomes made from synthetic lipids. We demonstrated production of ATP at steady-state conditions with the ATP synthase, driven by proton pumping by cytochrome bo3. Introduction of anionic lipids decreased the coupled activity and we could correlate this effect to weaker interactions between ATP synthase and cytochrome bo3 in the membrane. We also reconstituted cytochrome c oxidase (CytcO) from Saccharomyces cerevisiae with Respiratory supercomplex factor 1 (Rcf1) into liposomes and submitochondrial particles (SMPs). Loss of Rcf1 has previously been found to result in a lower CytcO activity. We found that activity could be restored upon co-reconstitution of CytcO with Rcf1, but only after unfolding and re-folding of the latter, which shows that Rcf1 can adopt two configurations in the membrane.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 67
Keywords
Cytochrome c oxidase, ATP synthase, Bioenergetics, membrane-mimetics, Rcf1, liposomes, oxidative phosphorylation, lipids, protein-protein interactions
National Category
Biochemistry and Molecular Biology Other Chemistry Topics
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-173617 (URN)978-91-7797-845-9 (ISBN)978-91-7797-846-6 (ISBN)
Public defence
2019-11-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

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

Available from: 2019-10-30 Created: 2019-09-26 Last updated: 2019-11-27Bibliographically approved

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