bims-mitran 2023-06-04 papers

Issue of 2023–06–04
three papers selected by
Andreas Kohler, University of Graz

FEBS Lett. 2023 May 29.

Cytochrome c oxidase biogenesis - from translation to early assembly of the core subunit COX1.

Sven Dennerlein, Peter Rehling, Ricarda Richter-Dennerlein.

Mitochondria are the powerhouses of the cell as they produce the majority of ATP with their oxidative phosphorylation (OXPHOS) machinery. The OXPHOS system is composed of the F1 Fo ATP synthase and four mitochondrial respiratory chain complexes, the terminal enzyme of which is the cytochrome c oxidase (complex IV) that transfers electrons to oxygen, generating water. Complex IV comprises of 14 structural subunits of dual genetic origin: while the three core subunits are mitochondrial encoded, the remaining constituents are encoded by the nuclear genome. Hence, the assembly of complex IV requires the coordination of two spatially separated gene expression machinery. Recent efforts elucidated an increasing number of proteins involved in mitochondrial gene expression, which are linked to complex IV assembly. Additionally, several COX1 biogenesis factors have been intensively biochemically investigated and an increasing number of structural snapshots shed light on the organization of macromolecular complexes such as the mitoribosome or the cytochrome c oxidase. Here, we focus on COX1 translation regulation and highlight the advanced understanding of early steps during COX1 assembly and its link to mitochondrial translation regulation.

Keywords: COX1; OXPHOS; complex IV; cytochrome c oxidase; mitochondria

DOI: https://doi.org/10.1002/1873-3468.14671

Bio Protoc. 2023 May 20. 13(10): e4680

Mitochondrial Replication Assay (MIRA) for Efficient in situ Quantification of Nascent mtDNA and Protein Interactions with Nascent mtDNA (mitoSIRF).

Macy Lozen, Yue Chen, Rebecca A Boisvert, Katharina Schlacher.

Mitochondria play decisive roles in bioenergetics and intracellular communication. These organelles contain a circular mitochondrial DNA (mtDNA) genome that is duplicated within one to two hours by a mitochondrial replisome, independently from the nuclear replisome. mtDNA stability is regulated in part at the level of mtDNA replication. Consequently, mutations in mitochondrial replisome components result in mtDNA instability and are associated with diverse disease phenotypes, including premature aging, aberrant cellular energetics, and developmental defects. The mechanisms ensuring mtDNA replication stability are not completely understood. Thus, there remains a need to develop tools to specifically and quantifiably examine mtDNA replication. To date, methods for labeling mtDNA have relied on prolonged exposures of 5'-bromo-2'-deoxyuridine (BrdU) or 5'-ethynyl-2'-deoxyuridine (EdU). However, labeling with these nucleoside analogs for a sufficiently short time in order to monitor nascent mtDNA replication, such as under two hours, does not produce signals suited for efficient or accurate quantitative analysis. The assay system described here, termed Mitochondrial Replication Assay (MIRA), utilizes proximity ligation assay (PLA) combined with EdU-coupled Click-IT chemistry to address this limitation, thereby enabling sensitive and quantitative analysis of nascent in situ mtDNA replication with single-cell resolution. This method can be further paired with conventional immunofluorescence (IF) for multi-parameter cell analysis. By enabling monitoring nascent mtDNA prior to the complete replication of the entire mtDNA genome, this new assay system allowed the discovery of a new mitochondrial stability pathway, mtDNA fork protection. Moreover, a modification in primary antibodies application allows the adaptation of our previously described in situ protein Interactions with nascent DNA Replication Forks (SIRF) for the detection of proteins of interest to nascent mtDNA replication forks on a single molecule level (mitoSIRF). Graphical overview Schematic overview of Mitochondrial Replication Assay (MIRA). 5'-ethynyl-2'-deoxyuridine (EdU; green) incorporated in DNA is tagged with biotin (blue) using Click-IT chemistry. Subsequent proximity ligation assay (PLA, pink circles) using antibodies against biotin allows the fluorescent tagging of the nascent EdU and amplification of the signal sufficient for visualization by standard immunofluorescence. PLA signals outside the nucleus denote mitochondrial DNA (mtDNA) signals. Ab, antibody. In in situ protein interactions with nascent DNA replication forks (mitoSIRF), one of the primary antibodies is directed against a protein of interest, while the other detects nascent biotinylated EdU, thus enabling in situ protein interactions with nascent mtDNA.

Keywords: BRCA2; Fanconi anemia; MIRA; Mitochondria; Mitochondrial DNA; Proximity ligation assay; mtDNA instability; mtDNA replication

DOI: https://doi.org/10.21769/BioProtoc.4680

Free Radic Biol Med. 2023 May 27. pii: S0891-5849(23)00446-X. [Epub ahead of print]

PET117 assembly factor stabilizes translation activator TACO1 thereby upregulates mitochondria-encoded cytochrome C oxidase 1 synthesis.

Qiong Sun, Le Shi, Shuaijun Li, Jialu Li, Ruifen Zhang, Xinghuai Huang, Yongping Shao, Zhihui Feng, Yunhua Peng, Zhiwei Yang, Jiankang Liu, Huadong Liu, Jiangang Long.

Cytochrome c oxidase, also known as complex IV, facilitates the transfer of electrons from cytochrome c to molecular oxygen, resulting in the production of ATP. The assembly of complex IV is a tightly regulated and intricate process that entails the coordinated synthesis and integration of subunits encoded by the mitochondria and nucleus into a functional complex. Accurate regulation of translation is crucial for maintaining proper mitochondrial function, and defects in this process can lead to a wide range of mitochondrial disorders and diseases. However, the mechanisms governing mRNA translation by mitoribosomes in mammals remain largely unknown. In this study, we elucidate the critical role of PET117, a chaperone protein involved in complex IV assembly, in the regulation of mitochondria-encoded cytochrome c oxidase 1 (COX1) protein synthesis in human cells. Depletion of PET117 reduced mitochondrial oxygen consumption rate and impaired mitochondrial function. PET117 was found to interact with and stabilize translational activator of COX1 (TACO1) and prevent its ubiquitination. TACO1 overexpression rescued the inhibitory effects on mitochondria caused by PET117 deficiency. These findings provide evidence for a novel PET117-TACO1 axis in the regulation of mitochondrial protein expression, and revealed a previously unknown role of PET117 in human cells.

Keywords: COX1; Mitochondrion; PET117; Protein stability; TACO1

DOI: https://doi.org/10.1016/j.freeradbiomed.2023.05.023