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Structural basis for Cbp3 interaction with newly synthesized cytochrome b during mitochondrial respiratory chain assembly
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bath, UK.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2019 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 294, no 45, p. 16663-16671Article in journal (Refereed) Published
Abstract [en]

Assembly of the mitochondrial respiratory chain requires the coordinated synthesis of mitochondrial and nuclear encoded subunits, redox co-factor acquisition, and correct joining of the subunits to form functional complexes. The conserved Cbp3–Cbp6 chaperone complex binds newly synthesized cytochrome b and supports the ordered acquisition of the heme co-factors. Moreover, it functions as a translational activator by interacting with the mitoribosome. Cbp3 consists of two distinct domains, an N-terminal domain present in mitochondrial Cbp3 homologs, and a highly conserved C-terminal domain comprising a ubiquinol–cytochrome c chaperone region. Here, we solved the crystal structure of this C-terminal domain from a bacterial homolog at 1.4 Å resolution, revealing a unique all-helical fold. This structure allowed mapping of the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b via site-specific photo-crosslinking. We propose that mitochondrial Cbp3 homologs carry an N-terminal extension that positions the conserved C-terminal domain at the ribosomal tunnel exit for an efficient interaction with its substrate, the newly synthesized cytochrome b protein.

Place, publisher, year, edition, pages
2019. Vol. 294, no 45, p. 16663-16671
Keywords [en]
respiratory chain, complex III, assembly factor, mitochondrial translation, protein assembly, membrane biogenesis, protein crosslinking, ubiquinol-cytochrome c chaperone domain, structural biology, electron transfer chain
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-171513DOI: 10.1074/jbc.RA119.010483OAI: oai:DiVA.org:su-171513DiVA, id: diva2:1342332
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-11-11Bibliographically approved
In thesis
1. Structure and Biogenesis of Membrane Proteins
Open this publication in new window or tab >>Structure and Biogenesis of Membrane Proteins
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins make up about one-third of the cellular proteome. The diverse roles that membrane proteins have in cells include major life-sustaining processes, making them major drug targets. The respiratory chain comprises a series of complexes of membrane proteins residing in the inner mitochondrial membrane, which serve as major drivers of ATP synthesis. Assembly of the respiratory chain complexes (RCC) requires coordinated synthesis of nuclear and mitochondrial subunits. Cbp3-Cbp6 complex binds to the mitoribosome as translational activator for cytochrome b synthesis and binds the nascent polypeptide to facilitate its hemylation. Cbp3 consists of an N-terminal domain specific to mitochondrial homologues and a conserved C-terminal ubiquinol-cytochrome c chaperone domain. In this thesis I present the first crystal structure of the C-terminal domain from a bacterial homologue that has enabled us to identify the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b using site-specific photo-crosslinking. Our finding suggests that Cbp3 contacts the mitoribosome via the N-terminal domain in a manner that positions the substrate binding site close to the tunnel exit. In the second project, we have analyzed the effects of disease causing cytochrome b mutations, on bc1 complex assembly. We found that complex III assembly is blocked at either intermediate 0 or I due to impaired insertion of bL or bH heme respectively, which indicates that assembly processes are involved in disease development. We then focused on NADH; a product of alpha-ketoglutarate dehydrogenase complex (KGDH) catalyzed citric acid cycle reaction and one of the substrates that supply electron to the respiratory chain. Kgd4 is a novel subunit of this enzyme complex and two functional variants (Kgd4S and Kgd4L) of unknown origins exist in yeast. We report in our work that Kgd4L originates from a UUG alternative start site, 90 nucleotides upstream and in frame of the annotated start codon. The sequence context upstream of UUG determines the efficiency of recognition of this alternative start codon. Finally, Na+/H+ antiporters are present in all species and are involved in regulation of intracellular pH, cell volume and sodium concentration. ATP formed during oxidative phosphorylation serves as energy source for Na+/K+ ATPase to generate Na+ gradient across the inner mitochondrial membrane, which drives local Na+/H+ antiporters. We show that K305 is involved in proton transport and responsible for the electrogenicity of NapA, while human NHA2 shows electroneutral antiporter activity.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 53
Keywords
Cbp3, cytochrome b, respiratory complex III, alternative translation initiation and sodium/proton exchange
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-171519 (URN)978-91-7797-749-0 (ISBN)978-91-7797-750-6 (ISBN)
Public defence
2019-09-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 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 2: Manuscript.

Available from: 2019-09-03 Created: 2019-08-13 Last updated: 2019-08-26Bibliographically approved
2. Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
Open this publication in new window or tab >>Mechanistic Insights in the Biogenesis and Function of the Respiratory Chain
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mitochondria fulfill a plethora of functions, including harboring metabolic pathways and converting energy stored in metabolites into ATP, the common energy source of the cell. This last function is performed by the oxidative phosphorylation system, consisting of the respiratory chain and the ATP synthase. Electrons are channeled through the complexes of the respiratory chain, while protons are translocated across the inner mitochondrial membrane. This process establishes an electrochemical gradient, which is used by the ATP synthase to generate ATP. The subunits of two of the respiratory chain complexes, the bc1 complex and the cytochrome c oxidase, are encoded by two genetic origins, the nuclear and the mitochondrial genome. Therefore, the assembly of these complexes needs to be coordinated and highly regulated.

Several proteins are involved in the biogenesis of the bc1 complex. Amongst these proteins, the Cbp3-Cbp6 complex was shown to regulate translation and assembly of the bc1 complex subunit cytochrome b. In this work, we established a homology model of yeast Cbp3. Using a site-specific crosslink approach, we identified binding sites of Cbp3 to its obligate binding partner Cbp6 and its client, cytochrome b, enabling a deeper insight in the molecular mechanisms of bc1 complex biogenesis. 

The bc1 complex and the cytochrome c oxidase form macromolecular structures, called supercomplexes. The detailed assembly mechanisms and functions of these structures remain to be solved. Two proteins, Rcf1 and Rcf2, were identified associating with supercomplexes in the yeast Saccharomyces cerevisiae. Our studies demonstrate that, while Rcf1 has a minor effect on supercomplex assembly, its main function is to modulate cytochrome c oxidase activity. We show that cytochrome c oxidase is present in three structurally different populations. Rcf1 is needed to maintain the dominant population in a functionally active state. In absence of Rcf1, the abundance of a population with an altered active site is increased. We propose that Rcf1 is needed, especially under a high work load of the respiratory chain, to maintain the function of cytochrome c oxidase.

This thesis aims to unravel molecular mechanisms of proteins involved in biogenesis and functionality of respiratory chain complexes to enable a deeper understanding. Dysfunctional respiratory chain complexes lead to severe disease, emphasizing the importance of this work.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 75
Keywords
respiratory chain, bc1 complex, cytochrome c oxidase, Cbp3, Rcf1, Rcf2, respiratory supercomplexes, biogenesis, mitochondria, Saccharomyces cerevisiae
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175276 (URN)978-91-7797-839-8 (ISBN)978-91-7797-840-4 (ISBN)
Public defence
2019-12-06, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2019-11-13 Created: 2019-10-16 Last updated: 2019-11-04Bibliographically approved

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