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Significance of mitochondrial ultrastructure for bioenergetics
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria are the site where most of the energy from food is converted into adenosine triphosphate (ATP). This process is taking place at the inner membrane (IM) of mitochondria, and is called oxidative phosphorylation, and results in the establishment of a proton motive force (pmf). The proton motive force is a combination of a proton difference over the mitochondrial IM and a charge difference. The ATP is then synthesized by the ATP synthase, which is utilizing the pmf for this process. The IM of mitochondria has many invaginations, which are called cristae. The enzymes of the respiratory chain are mainly located at the flat sheet part, while the ATP synthase is located at the rim of the cristae. The hypothesis arises whether the cristae membrane would serve as a proton sink for the ATP synthase, due to the curved shape of the cristae. We aimed at answering this hypothesis by attaching a pH-sensitive green fluorescent protein (pHluorin2) at different locations within the mitochondria. The study revealed that there is no substantial pH difference across the IM of yeast mitochondria and that the cristae are not functioning as a proton sink, but rather its main function is to provide an optimal environment, for coupled enzymatic activity. The second project investigated the importance of the mobile electron carrier; cytochrome c (cyt c) of its ability to freely diffuse along with the IM. Cyt c is the electron carrier between the bc1 complex and cytochrome c oxidase of the respiratory chain. It is also involved in programmed cell death (apoptosis) of higher eukaryotes, where its release from mitochondria initiates apoptosis. As its role in yeast apoptosis is not entirely clear, we created a yeast strain where cyt c was tethered to the IM, in a background strain that was devoid of the mobile cyt c. Interestingly, the level of apoptosis was higher in the yeast strain with the non-mobile cyt c, which indicated that cyt c release in yeast is not a necessary step to initiate apoptosis. The strain with the IM tethered cyt c had also higher levels of reactive oxygen species (ROS), shorter life span, alterations of the mitochondrial network in comparison to the wild type strain. Despite not showing any major alterations in the respiratory chain, the mutant yeast strain had elevated oxygen consumption, indicating a compensatory mechanism, which could have caused the elevated ROS levels which ultimately induced apoptosis. Maintaining a steady level of ATP is crucial for the cell, and one such mechanism in higher eukaryotes is the creatine phosphate shuttle system, by the enzyme; creatine kinase. Creatine kinase is catalyzing the phosphorylation of creatine in mitochondria, and the phosphocreatine is transported out to the cytosol, where the cytoplasmic isoform of the enzyme is regenerating ATP from the phosphocreatine. A yeast strain was created to express the mitochondrial creatine kinase, which could serve as a strategy in industrially relevant yeast strains, to circumvent ATP levels to drop during the production processes. To gain an understanding of the importance of cyt c diffusion, its relevance for the respiratory chain, the yeast strain from the second project was modified so that it was fused to the bc1 complex. The strain showed a functional respiratory chain, and further work will provide insights into the diffusion of the respiratory complexes and their interaction with the IM.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2020. , p. 75
Keywords [en]
bioenergetics, mitochondrial ultrastructure, respiratory chain, Saccharomyces cereviciae, ATP synthase, apoptosis, cytochrome c, supercomplexes
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-180530ISBN: 978-91-7911-118-2 (print)ISBN: 978-91-7911-119-9 (electronic)OAI: oai:DiVA.org:su-180530DiVA, id: diva2:1420820
Public defence
2020-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Submitted. Paper 4: Manuscript.

Available from: 2020-05-05 Created: 2020-03-31 Last updated: 2020-05-26Bibliographically approved
List of papers
1. Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes
Open this publication in new window or tab >>Kinetic coupling of the respiratory chain with ATP synthase, but not proton gradients, drives ATP production in cristae membranes
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2020 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 117, no 5, p. 2412-2421Article in journal (Refereed) Published
Abstract [en]

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.

Keywords
mitochondria, ATP synthesis, cristae, energy conversion, kinetic coupling
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-179519 (URN)10.1073/pnas.1917968117 (DOI)000512340900032 ()31964824 (PubMedID)
Available from: 2020-03-09 Created: 2020-03-09 Last updated: 2020-04-14Bibliographically approved
2. Membrane tethering of cytochrome c accelerates apoptotic cell death in yeast
Open this publication in new window or tab >>Membrane tethering of cytochrome c accelerates apoptotic cell death in yeast
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2020 (English)In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889Article in journal (Refereed) Accepted
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180540 (URN)
Available from: 2020-04-01 Created: 2020-04-01 Last updated: 2020-04-14
3. Expression of human ubiquitous mitochondrial creatine kinase in yeast as a strategy for ATP-depletion recovery in commercial yeast strains
Open this publication in new window or tab >>Expression of human ubiquitous mitochondrial creatine kinase in yeast as a strategy for ATP-depletion recovery in commercial yeast strains
(English)In: Article in journal (Refereed) Submitted
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180541 (URN)
Available from: 2020-04-01 Created: 2020-04-01 Last updated: 2020-04-14Bibliographically approved
4. Fusing cytochrome c to cytochrome c1 of the bc1 complex maintains respiration in Saccharomyces cerevisiae
Open this publication in new window or tab >>Fusing cytochrome c to cytochrome c1 of the bc1 complex maintains respiration in Saccharomyces cerevisiae
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-180542 (URN)
Available from: 2020-04-01 Created: 2020-04-01 Last updated: 2020-04-14Bibliographically approved

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