Investigating the role of three proteins in mitochondrial homeostasis

Jose Gonzalez De Cozar

Research output: Book/ReportDoctoral thesisCollection of Articles

Abstract

The mitochondrion is commonly described as the powerplant of the eukaryotic cell, due to its unique role in energy transactions; although it is also a required organelle for metabolism, apoptosis, and thermostasis. The thirty-seven genes required to synthesise the bioeneregtic machinery are encoded in a small double-stranded mitochondrial genome (mtDNA). Loss or misregulation of mtDNA causes a set of devastating diseases such as MELAS or Alpers syndrome, with no currently available treatment. To elucidate the underlying molecular causes of these diseases it is essential to understand the mechanisms involved in mtDNA maintenance. Previously, a genome-wide screening for genes involved in mtDNA metabolism identified several novel or previously unsuspected proteins (Fukuoh et al., 2014), including subunits of the ATP synthase complex, mitochondrial biogenesis factors, and vesicle trafficking proteins. The research reported in this thesis aimed to broaden our understanding of the mitochondrial function(s) of a subset of these proteins, using Drosophila melanogaster as a model organism, applying a combination of cellular, genetic and biochemical techniques.

Two components of the vesicle-trafficking syntaxin 5-SNARE complex, Bet1 and Slh, were identified in the original screen. A detailed analysis found that downregulation (KD) of Slh and Bet1 did not affect mtDNA quantity or quality, but led to mitochondrial dysfunction by impairing respiration, mitochondrial content, membrane potential, and upregulation of the mitochondrial proteotoxic stress response machinery. The subcellular distribution of both proteins was investigated by immunocytochemistry and by Western blot analysis of subcellular fractions, revealing colocalization of Bet1 and Slh with the Golgi apparatus and late endosomes, whilst part of the Bet1 signal was also present in the mitochondrial fraction. Taken together with other findings from the laboratory, I propose that endoplasmic reticulum- Golgi vesicle sorting is involved in the mitochondrial quality control machinery.

Ribonuclease H1 (RNase H1, encoded in Drosophila by the rnh1 gene), an enzyme that degrades RNA/DNA hybrids, was also identified in the genome-wide screening. The expression of rnh1 was studied in in Drosophila S2 cells, revealing a heterogeneous distribution of the protein between mitochondria and nucleus. Based on these findings, the presence of two alternative translation initiation sites in a single messenger RNA is proposed to generate two alternative polypeptides distributing the enzyme between the two compartments. Rnh1 depletion in S2 cells was found to decrease mtDNA copy number without any impact on cell viability. On the contrary, low levels of rnh1 in flies led to features similar to those seen in some patients carrying mutations in RNASEH1, causing a decrease in lifespan and triggering severe locomotor dysfunction, accompanied by defects in mitochondrial respiration, DNA replication, and transcription. Modulation of rnh1 expression had systematic effects on mtDNA replication intermediates, especially in regions where transcription and DNA replication are inferred to progress in opposite directions. In addition, long-term rnh1 knockdown in S2 cells triggered the accumulation of four-way junctions and other topological abnormalities in mtDNA, as well as of structures associated with fork regression at the origin of replication. Overall, a tight connection between transcription and replication of the mtDNA is implied, with RNase H1 acting as a regulatory component, influencing both processes.

The loss of RNase H1 activity in the fly resulted in particular defects at the molecular level that were not seen in mammals, such as the appearance of abnormal four-way junctions, the accumulation of DNA replication intermediates in specific regions of the mtDNA or large changes in the relative abundance of different mitochondrial mRNAs. In part, this may reflect the differences in mtDNA organization and replication between mammals and Drosophila. To understand whether these differences are also caused by the unique biochemical properties of Drosophila RNase H1, I used a set of biochemical techniques to characterize the Drosophila enzyme and compared it to its human homolog. The canonical structure described for eukaryotic RNase H1 is also predicted to be present in the fly enzyme. A nucleic acid-binding element, denoted as the hybrid binding domain (HBD), is located in the N-terminal region, whilst the C-terminal region contains the catalytic domain; the two are connected through a linker without defined function or structure. The Drosophila linker is double the size of the human one. Structure modeling in silico predicted that it should adopt an HBD-like structure. Biochemical analysis of a deletion series of Drosophila RNase H1 variants revealed that this second HBD-like domain accelerates catalysis but weakens binding of the enzyme to RNA/DNA hybrid. I propose that these features of the second HBD confer processivity on the enzyme. A shotgun proteomic screen to identify RNase H1- interacting proteins yielded components of the mitochondrial and nuclear genome maintenance machinery, as well as several metabolic enzymes, translation factors and elements of the import and processing machinery. These included the mitochondrial single-stranded DNA binding protein (mtSSB) prompted me to conduct a closer analysis of its interaction with RNase H1, using in vivo and in vitro approaches. These experiments were unable to document any direct physical interaction between the two proteins, and their functional interaction was also negligible.

In conclusion, this project shed light on the direct or indirect mitochondrial roles of Bet1, Slh, and RNase H1, and investigated the unique biochemical properties of the Drosophila ribonuclease. Altogether, the findings underline that even though the scientific community has investigated mitochondria for more than a century, our understanding of mitochondrial functions and requirements are still far from complete. Further investigation is necessary to elucidate the pathways that converge in mitochondria to maintain the metabolic and energetic requirements of cells.
Original languageEnglish
Place of PublicationTampere
PublisherTampere University
ISBN (Electronic)978-952-03-1955-7
ISBN (Print)978-952-03-1954-0
Publication statusPublished - 2021
Publication typeG5 Doctoral dissertation (article)

Publication series

NameTampere University Dissertations - Tampereen yliopiston väitöskirjat
Volume416
ISSN (Print)2489-9860
ISSN (Electronic)2490-0028

Fingerprint

Dive into the research topics of 'Investigating the role of three proteins in mitochondrial homeostasis'. Together they form a unique fingerprint.

Cite this