scholarly journals The yeast protein Mam33 functions in the assembly of the mitochondrial ribosome

2019 ◽  
Vol 294 (25) ◽  
pp. 9813-9829 ◽  
Author(s):  
Gabrielle A. Hillman ◽  
Michael F. Henry
Keyword(s):  
Ribosomal Proteins ◽  
Matrix Protein ◽  
Synthetic Lethality ◽  
Large Subunit ◽  
Binding Assays ◽  
Safe Delivery ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Evolutionarily Conserved

Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. Although remarkable progress has been made toward understanding the structure of mitoribosomes, the pathways and factors that facilitate their biogenesis remain largely unknown. The long unstructured domains of unassembled ribosomal proteins are highly prone to misfolding and often require dedicated chaperones to prevent aggregation. To date, chaperones that ensure safe delivery to the assembling ribosome have not been identified in the mitochondrion. In this study, a respiratory synthetic lethality screen revealed a role for an evolutionarily conserved mitochondrial matrix protein called Mam33 in Saccharomyces cerevisiae mitoribosome biogenesis. We found that the absence of Mam33 results in misassembled, aggregated ribosomes and a respiratory lethal phenotype in combination with other ribosome-assembly mutants. Using sucrose gradient sedimentation, native affinity purifications, in vitro binding assays, and SILAC-based quantitative proteomics, we found that Mam33 does not associate with the mature mitoribosome, but directly binds a subset of unassembled large subunit proteins. Based on these data, we propose that Mam33 binds specific mitoribosomal proteins to ensure proper assembly.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals Mitochondrial Ribosome as the Target for the Macrolide Antibiotic Clarithromycin in the Helminth Echinococcus multilocularis

2005 ◽  
Vol 49 (8) ◽  
pp. 3251-3255 ◽  
Author(s):  
Alexander Mathis ◽  
Peter Wild ◽  
Erik C. Boettger ◽  
Christian M. O. Kapel ◽  
Peter Deplazes
Keyword(s):  
Echinococcus Multilocularis ◽  
Morphological Changes ◽  
Large Subunit ◽  
Dependent Manner ◽  
Morphological Alterations ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Effects ◽  
Large Subunit Rrna

ABSTRACT The mitochondrial rRNA of the tapeworm species Echinococcus multilocularis carries an adenine at sequence position 2058 (numbering according to that for Escherichia coli) of the large-subunit rRNA (lsrRNA), while the nucleus-encoded rRNA, as determined in this study, is characterized by 2058G. This indicates a dichotomy in the drug susceptibilities of ribosomes: cytoplasmic ribosomes are predicted to be resistant to macrolide antibiotics, while mitochondrial ribosomes lack the most common chromosomal resistance determinant, lsrRNA 2058G. Upon incubation with the macrolide clarithromycin, the formation of vesicles from metacestode tissue was reduced in a dose-dependent manner. Electron microscopy revealed distinct morphological alterations both of the mitochondria and of the vesicle wall (e.g., loss of microtriches) in drug-treated vesicles. Adult worms lost their motility and displayed morphological changes (shortening and constriction of proglottids and the presence of vacuoles) upon incubation with clarithromycin. Our findings demonstrate that macrolides have distinct in vitro effects on E. multilocularis, endorsing the use of sequence-based in silico approaches for exploitation of available ribosomal drugs as anthelmintic agents.

scholarly journals Abnormal Expression of Mitochondrial Ribosomal Proteins and Their Encoding Genes with Cell Apoptosis and Diseases

2020 ◽  
Vol 21 (22) ◽  
pp. 8879
Author(s):  
Guomin Huang ◽  
Hongyan Li ◽  
Hong Zhang
Keyword(s):  
Cell Apoptosis ◽  
Ribosomal Proteins ◽  
Mitochondrial Respiratory Chain ◽  
Basic Structure ◽  
Disease Diagnosis ◽  
Mitochondrial Ribosomes ◽  
Abnormal Expression ◽  
Metabolism Disorder ◽  
Mitochondrial Ribosome ◽  
Inducing Factors

Mammalian mitochondrial ribosomes translate 13 proteins encoded by mitochondrial genes, all of which play roles in the mitochondrial respiratory chain. After a long period of reconstruction, mitochondrial ribosomes are the most protein-rich ribosomes. Mitochondrial ribosomal proteins (MRPs) are encoded by nuclear genes, synthesized in the cytoplasm and then, transported to the mitochondria to be assembled into mitochondrial ribosomes. MRPs not only play a role in mitochondrial oxidative phosphorylation (OXPHOS). Moreover, they participate in the regulation of cell state as apoptosis inducing factors. Abnormal expressions of MRPs will lead to mitochondrial metabolism disorder, cell dysfunction, etc. Many researches have demonstrated the abnormal expression of MRPs in various tumors. This paper reviews the basic structure of mitochondrial ribosome, focuses on the structure and function of MRPs, and their relationships with cell apoptosis and diseases. It provides a reference for the study of the function of MRPs and the disease diagnosis and treatment.

scholarly journals A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sabina Schütz ◽  
Ute Fischer ◽  
Martin Altvater ◽  
Purnima Nerurkar ◽  
Cohue Peña ◽  
...  
Keyword(s):  
Yeast Cell ◽  
Nuclear Import ◽  
Generation Time ◽  
Ribosomal Proteins ◽  
Ribosome Assembly ◽  
Single Generation ◽  
Safe Delivery ◽  
Independent Mechanism ◽  
Assembly Site

Within a single generation time a growing yeast cell imports ∼14 million ribosomal proteins (r-proteins) into the nucleus for ribosome production. After import, it is unclear how these intrinsically unstable and aggregation-prone proteins are targeted to the ribosome assembly site in the nucleolus. Here, we report the discovery of a conserved nuclear carrier Tsr2 that coordinates transfer of the r-protein eS26 to the earliest assembling pre-ribosome, the 90S. In vitro studies revealed that Tsr2 efficiently dissociates importin:eS26 complexes via an atypical RanGTP-independent mechanism that terminates the import process. Subsequently, Tsr2 binds the released eS26, shields it from proteolysis, and ensures its safe delivery to the 90S pre-ribosome. We anticipate similar carriers—termed here escortins—to securely connect the nuclear import machinery with pathways that deposit r-proteins onto developing pre-ribosomal particles.

scholarly journals Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

2020 ◽  
Vol 117 (47) ◽  
pp. 29851-29861 ◽  
Author(s):  
Heddy Soufari ◽  
Florent Waltz ◽  
Camila Parrot ◽  
Stéphanie Durrieu-Gaillard ◽  
Anthony Bochler ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Large Subunit ◽  
Microscopy Study ◽  
Small Subunit ◽  
Electron Microscopy Study ◽  
Mitochondrial Ribosomes ◽  
Cryo Electron Microscopy ◽  
Parasite Survival ◽  
Assembly Intermediate ◽  
Early Translation

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

scholarly journals HybF, a Zinc-Containing Protein Involved in NiFe Hydrogenase Maturation

2004 ◽  
Vol 186 (9) ◽  
pp. 2603-2611 ◽  
Author(s):  
Melanie Blokesch ◽  
Michaela Rohrmoser ◽  
Sabine Rode ◽  
August Böck
Keyword(s):  
Large Subunit ◽  
Gel Filtration ◽  
Stoichiometric Ratio ◽  
Amino Acid Residues ◽  
Binding Assays ◽  
Triple Mutant ◽  
Hydrogenase Maturation ◽  
Identify Amino Acid

ABSTRACT HypA and HypB are maturation proteins required for incorporation of nickel into the hydrogenase large subunit. To examine the functions of these proteins in nickel insertion, the hybF gene, which is a homolog of hypA essential for maturation of hydrogenases 1 and 2 from Escherichia coli, was overexpressed, and the product was purified. This protein behaves like a monomer in gel filtration and contains stoichiometric amounts of zinc but insignificant or undetectable amounts of nickel and iron. In filter binding assays radioactively labeled nickel binds to HybF with a KD of 1.87 μM and in a stoichiometric ratio. To identify amino acid residues of HybF involved in nickel and/or zinc binding, variants in which conserved residues were replaced were studied. An H2Q replacement eliminated both in vivo activity and in vitro binding of nickel. The purified protein, however, contained zinc at the level characteristic of the wild-type protein. When E3 was replaced by Q, activity was retained, but an E3L exchange was detrimental. Replacement of each of the four conserved cysteine residues of a zinc finger motif reduced the cellular amount of HybF protein without a loss of in vivo activity, indicating that these residues play a purely structural role. A triple mutant deficient in the synthesis or activity of HypA, HybF, and HypB was constructed, and it exhibited the same responsiveness for phenotypic complementation by high nickel as mutants with a single lesion in one of the genes exhibited. The results are interpreted in terms of a concerted action of HypB and HybF in nickel insertion in which HybF (as well as its homolog, HypA) functions as a metallochaperone and HypB functions as a regulator that controls the interaction of HybF with the target protein.

scholarly journals Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins

2021 ◽  
Vol 22 (11) ◽  
pp. 5496
Author(s):  
Annalisa Pecoraro ◽  
Martina Pagano ◽  
Giulia Russo ◽  
Annapina Russo
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Protein Biosynthesis ◽  
Cellular Respiration ◽  
Mitochondrial Ribosomes ◽  
Ribonucleoprotein Complexes ◽  
Mitochondrial Ribosome ◽  
Energy Consuming ◽  
Cell Growth And Proliferation

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

scholarly journals Interactions of Pex7p and Pex18p/Pex21p with the Peroxisomal Docking Machinery: Implications for the First Steps in PTS2 Protein Import

2002 ◽  
Vol 22 (17) ◽  
pp. 6056-6069 ◽  
Author(s):  
Katharina Stein ◽  
Annette Schell-Steven ◽  
Ralf Erdmann ◽  
Hanspeter Rottensteiner
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Dependent Manner ◽  
Binding Assays ◽  
Interaction Domain ◽  
Vitro Binding ◽  
Targeting Signal ◽  
Two Hybrid ◽  
Import Receptor

ABSTRACT Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13Δ mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.

scholarly journals Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Victor Tobiasson ◽  
Alexey Amunts
Keyword(s):  
Mitochondrial Genome ◽  
Ribosomal Proteins ◽  
Protein Targeting ◽  
Large Subunit ◽  
Model Organism ◽  
Small Subunit ◽  
Specific Protein ◽  
Mitochondrial Translation ◽  
Functional Protein ◽  
Mitochondrial Ribosome

To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

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