Session 1: Mitochondrial architecture and behavior (contd.)

The mitoribosome translates specific mitochondrial mRNAs and regulates energy production that is a signature of all eukaryotic life forms. We present cryo-EM analyses of its assembly intermediates, mRNA binding process, and nascent polypeptide delivery to the membrane. To study the assembly mechanism, we determined a series of the small mitoribosomal subunit intermediates in complex with auxiliary factors that explain how action of step-specific factors establishes the catalytic mitoribosome. It features a mitochondria-specific protein ms37 that links the assembly to translation initiation. A delivery of mRNA is then performed by a 150kDa protein LRPPRC that forms a stable complex with a small binding partner SLIRP. In mammals, LRPPRC stabilises mRNAs co-transcriptionally, thus it links the entire gene expression system. Specific mitoribosomal proteins align the delivered mRNA with tRNA in the decoding center. Through the translation cycle, a nascent polypeptide is delivered to the mitochondrial inner membrane, and we report the mitoribosome structure bound to the insertase OXA1, which elucidates the basis for protein synthesis coupling to membrane delivery. Finally, comparative structural and biochemical analyses reveal functionally important binding of cofactors NAD, ATP, GDP, iron-sulfur clusters and polyamines. Together with experimental identification of specific rRNA and protein modifications, the data illuminate principal components responsible for the translation of genetic material in mitochondria.

References:

Activation mechanism of mitochondrial translation by LRPPRC-SLIRP. bioRxiv (2022). Singh V, Itoh Y, Huynen M & Amunts A.

Mechanism of mitoribosomal small subunit biogenesis and preinitiation. Nature (2022). Itoh Y, Khawaja A, Laptev I, Cipullo M, Atanassov I, Sergiev P, Rorbach J & Amunts A. 

Mechanism of membrane-tethered mitochondrial protein synthesis. Science (2021).
 Itoh Y, Andrell J, Choi A, Richter U, Maiti P, Best R, Barrientos A, Battersby B & Amunts A.

The electron-transport-chain/ETC functions at the Inner Mitochondrial Membrane (IMM) relies on mobile electron-carriers. The lateral diffusion of mobile carriers within IMM has long been proposed to impact ETC flux/respiration rates. From this viewpoint, IMM fluidity would be an important factor regulating respiration rates and overall cellular physiology. However, the role of fluidity in regulating ETC/respiratory flux has often been debated, especially with the discovery of ETC 'supercomplexes'. What is also unclear is if cells rapidly change IMM fluidity in response to stimuli and overall cell state. Despite contrasting views, there is increasing recognition that fluidity changes can regulate respiration and the overall cellular physiology. For instance, using an engineered E.coli system amenable to precise control, Budin et al Science 2018 showed that membrane viscosity influences rates of respiration. Intriguingly, Jose Enrique, Martinez-Ruiz-et-al (Nature 2020) find IMM-fluidity to regulate ROS in hypoxia. IMM fluidity may play differential roles depending on the precise electron donor (FADH versus NADH) feeding into ETC. Despite clear importance, information on IMM fluidity from intact, living-cells is severely lacking.  Here we present a robust, reliable, fully-validated, method/tool for accurately mapping IMM fluidity in living cells with spatio-temporal precision. Method uses a highly sensitive, photostable/low-toxicity, red-shifted, cell-permeable fluorescence-lifetime/FLIM probe that is easily used all cells and tissues. In this imaging method, we combine accurate IMM fluidity measurement with overall ratio of glycolysis/respiration through NADH autofluorescence-lifetimes at a single cell level. We find that a tremendous heterogeneity in IMM ordering/fluidity even within a single mitochondria. Notably we find that IMM fluidity gets dynamically and rapidly modulated in response to  multiple stimuli including overall state of respiration. This method/tools opens new avenues of inquiry and can clarify IMM dynamics and adaptation in a variety of cellular processes.

Maintaining mitochondrial proteostasis and function are key for cellular survival. How mitochondrial protein (mito-protein) misfolding activates mitophagy and communicates with cellular stress responses remains unclear. Carrying out a genome-wide CRISPR/Cas9 screen, we identified that reducing mito-protein import was sufficient to induce mitophagy without a need for depolarization. Upon mito-protein misfolding, the PAM complex, an essential part of the mito-protein import machinery, moves from the translocon to the insoluble mito-protein fraction. Thus, it titrates its mito-protein import and folding functions to adjust import rates to the folding status. When mito-protein folding is perturbed, the resulting reduction in import drives mitophagy activation to remove dysfunctional mitochondria. Employing a novel mito-protein import proteomics assay, we defined specific import changes and its links to cellular stress response systems. Together, our findings reveal the mechanism how mito-protein misfolding induces mitophagy and provide first insight into the complex mito-protein import rate changes during mitochondrial stress.

The tumor suppressor gene PTEN is the second most commonly mutated gene in human cancer. Genomic deletions of PTEN frequently include the adjacent gene, ATAD1. Previous work established that ATAD1 prevents the accumulation of proteins on the outer mitochondrial membrane, is an essential gene in humans and mice, and is conserved across eukaryotes. We used genome-wide CRISPR screens to identify vulnerabilities that exist specifically in cells that lack ATAD1, since these could distinguish tumor cells from host cells in select cancer patients.

We report that co-deletion of ATAD1 along with PTEN makes cancer cells sensitive to clinically-used inhibitors of the proteasome. When the ubiquitin-proteasome system is disrupted, pro-death proteins localize to mitochondria to trigger apoptosis. In healthy cells, ATAD1 directly and specifically extracts these proteins to remove them from mitochondria and prevent cell death. Hence, cancer cells that coincidentally co-delete ATAD1 along with PTEN lack the protective effects of ATAD1 and are consequently hypersensitive to proteasome inhibition. Indeed, proteasome inhibitors significantly decrease the growth of mouse xenograft tumors that lack ATAD1, but have no effect on isogenic tumors with functional ATAD1. ATAD1/PTEN co-deletion occurs in up to 33% of metastatic prostate cancer, 8% of glioblastoma, and 11% of melanoma, so the scope of potential impact is considerable. Our findings teach us about how eukaryotic cells cope with protein stress and identify an actionable therapeutic opportunity for hundreds of thousands of cancer patients.

We are interested in understanding the unique mechanisms of protein synthesis that take place in mitochondria and the biogenesis of mitochondrial ribosomes.  To this account we determined the unusual structure of mammalian mitochondrial ribosomes and revealed many unique aspects of translation initiation, elongation and termination. These results explained, for example, the mechanism of tRNA selection by the unusual mitochondrial initiation factor 2, revealed the mechanism of how mitochondrial ribosomes co-translationally target proteins to the inner mitochondrial membrane, and how leaderless mitochondrial mRNAs are threaded into the mRNA channel. We also investigated the biogenesis of mitochondrial ribosomes in mammalian cells and in trypanosomes, parasitic protozoans that cause a range of diseases. Our studies revealed the sequential order of events involving numerous ribosome biogenesis factors that govern the maturation of human mitochondrial ribosomes and the assembly of trypanosomal mitoribosomes. These results provide the basis for future structural, biochemical, genetic and cellular investigation of mitochondrial protein synthesis and the associated cellular machinery.

Kummer E, Leibundgut M, Rackham O, Lee RG, Boehringer D, Filipovska A, Ban N. (2018) Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature. 560(7717):263-267.

Saurer M., et al. and Schneider A, Ban N. (2020) Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery. Science 365 (6458), 1144-1149

Lenarčič T., et al and Rackham O., Filipovska A., & Ban N. (2021). Stepwise maturation of the peptidyl transferase region of human mitoribosomes. Nature Communications, 12(1), 3671.

Kummer E., Schubert K.N., Schoenhut T., Scaiola A., Ban N. (2021) Structural basis of translation termination, rescue, and recycling in mammalian mitochondria. Mol Cell. 2021 Jun 17;81(12):2566-2582

Lenarčič T, et al. and Schneider A and Ban N. (2022) Mitoribosomal small subunit maturation involves formation of initiation-like complexes. Proc Nat Acad Sci USA 119 (3) e2114710118