scholarly journals The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

2018 ◽  
Vol 29 (20) ◽  
pp. 2386-2396 ◽  
Author(s):  
Braulio Vargas Möller-Hergt ◽  
Andreas Carlström ◽  
Katharina Stephan ◽  
Axel Imhof ◽  
Martin Ott
Keyword(s):  
Membrane Protein ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Mitochondrial Translation ◽  
Protein Insertion ◽  
Mitochondrial Ribosomes ◽  
Protein Biogenesis ◽  
Membrane Bound ◽  
Highly Hydrophobic ◽  
Soluble C

Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for the production of highly hydrophobic subunits of the oxidative phosphorylation system. Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria.

Membrane protein import in yeast mitochondria

2000 ◽  
Vol 28 (4) ◽  
pp. 495-499 ◽  
Author(s):  
K. Tokatlidis ◽  
S. Vial ◽  
P. Luciano ◽  
M. Vergnolle ◽  
S. Clémence
Keyword(s):  
Membrane Protein ◽  
Molecular Mechanism ◽  
Protein Import ◽  
Mitochondrial Matrix ◽  
Yeast Mitochondria ◽  
Inner Membrane ◽  
Protein Insertion ◽  
The Past ◽  
Membrane Protein Insertion ◽  
Membrane Bound

The protein import pathway that targets proteins to the mitochondrial matrix has been extensively characterized in the past 15 years. Variations of this import pathway account for the sorting of proteins to other compartments as well, but the insertion of integral inner membrane proteins lacking a presequence is mediated by distinct translocation machinery. This consists of a complex of Tim9 and Tim10, two homologous, Zn2+-binding proteins that chaperone the passage of the hydrophobic precursor across the aqueous inter-membrane space. The precursor is then targeted to another, inner-membrane-bound, complex of at least five subunits that facilitates insertion. Biochemical and genetic experiments have identified the key components of this process; we are now starting to understand the molecular mechanism. This review highlights recent advances in this new membrane protein insertion pathway.

scholarly journals A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

1990 ◽  
Vol 10 (9) ◽  
pp. 4590-4595 ◽  
Author(s):  
T W McMullin ◽  
P Haffter ◽  
T D Fox
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Nuclear Gene ◽  
Small Subunit ◽  
Rrna Gene ◽  
Mitochondrial Translation ◽  
Activator Proteins ◽  
Translational Activator

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.

scholarly journals The architecture of EMC reveals a path for membrane protein insertion

2020 ◽  
Author(s):  
John P. O’Donnell ◽  
Ben P. Phillips ◽  
Yuichi Yagita ◽  
Szymon Juszkiewicz ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Integral Membrane Proteins ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Chaperone Function ◽  
Eukaryotic Genes ◽  
Biochemical Assays ◽  
Protein Biogenesis ◽  
Membrane Protein Insertion

AbstractApproximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function.

scholarly journals Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion

2021 ◽  
Vol 220 (11) ◽  
Author(s):  
Un Seng Chio ◽  
Yumeng Liu ◽  
SangYoon Chung ◽  
Woo Jun Shim ◽  
Sowmya Chandrasekar ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Eukaryotic Cells ◽  
Protein Insertion ◽  
Protein Biogenesis ◽  
Cytosolic Domains ◽  
Membrane Protein Insertion ◽  
The Individual ◽  
Molecular Recognition Features

The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.

scholarly journals The architecture of EMC reveals a path for membrane protein insertion

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
John P O'Donnell ◽  
Ben P Phillips ◽  
Yuichi Yagita ◽  
Szymon Juszkiewicz ◽  
Armin Wagner ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Transmembrane Domain ◽  
Integral Membrane Proteins ◽  
Membrane Insertion ◽  
Protein Insertion ◽  
Chaperone Function ◽  
Eukaryotic Genes ◽  
Biochemical Assays ◽  
Protein Biogenesis ◽  
Membrane Protein Insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC’s cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate’s transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC’s proposed chaperone function.

scholarly journals Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

2020 ◽  
Vol 48 (22) ◽  
pp. 12929-12942 ◽  
Author(s):  
Elena Lavdovskaia ◽  
Kärt Denks ◽  
Franziska Nadler ◽  
Emely Steube ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Dual Function ◽  
Large Ribosomal Subunit ◽  
Ribosome Recycling Factor ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Assembly Intermediate

Abstract Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

scholarly journals A stalled-ribosome rescue factor Pth3 is required for mitochondrial translation against antibiotics in Saccharomyces cerevisiae

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Soichiro Hoshino ◽  
Ryohei Kanemura ◽  
Daisuke Kurita ◽  
Yukihiro Soutome ◽  
Hyouta Himeno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stop Codon ◽  
Mitochondrial Gene ◽  
Mrna Levels ◽  
Primary Role ◽  
Mitochondrial Gene Expression ◽  
Rt Pcr ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
In Organello

AbstractMitochondrial translation appears to involve two stalled-ribosome rescue factors (srRFs). One srRF is an ICT1 protein from humans that rescues a “non-stop” type of mitochondrial ribosomes (mitoribosomes) stalled on mRNA lacking a stop codon, while the other, C12orf65, reportedly has functions that overlap with those of ICT1; however, its primary role remains unclear. We herein demonstrated that the Saccharomyces cerevisiae homolog of C12orf65, Pth3 (Rso55), preferentially rescued antibiotic-dependent stalled mitoribosomes, which appear to represent a “no-go” type of ribosomes stalled on intact mRNA. On media containing a non-fermentable carbon source, which requires mitochondrial gene expression, respiratory growth was impaired significantly more by the deletion of PTH3 than that of the ICT1 homolog PTH4 in the presence of antibiotics that inhibit mitochondrial translation, such as tetracyclines and macrolides. Additionally, the in organello labeling of mitochondrial translation products and quantification of mRNA levels by quantitative RT-PCR suggested that in the presence of tetracycline, the deletion of PTH3, but not PTH4, reduced the protein expression of all eight mtDNA-encoded genes at the post-transcriptional or translational level. These results indicate that Pth3 can function as a mitochondrial srRF specific for ribosomes stalled by antibiotics and plays a role in antibiotic resistance in fungi.

scholarly journals YBEY is an essential biogenesis factor for mitochondrial ribosomes

2019 ◽  
Author(s):  
Sabrina Summer ◽  
Anna Smirnova ◽  
Alessandro Gabriele ◽  
Ursula Toth ◽  
Fasemore Mandela ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ribosome Biogenesis ◽  
Specific Interaction ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Abnormal Mitochondria

ABSTRACTRibosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3’-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter recruits uS11m to the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

scholarly journals YBEY is an essential biogenesis factor for mitochondrial ribosomes

2020 ◽  
Vol 48 (17) ◽  
pp. 9762-9786 ◽  
Author(s):  
Sabrina Summer ◽  
Anna Smirnova ◽  
Alessandro Gabriele ◽  
Ursula Toth ◽  
Akinyemi Mandela Fasemore ◽  
...  
Keyword(s):  
16S Rrna ◽  
Ribosome Biogenesis ◽  
Specific Interaction ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
12S Rrna ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Abnormal Mitochondria

Abstract Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3′-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator

1990 ◽  
Vol 10 (9) ◽  
pp. 4590-4595
Author(s):  
T W McMullin ◽  
P Haffter ◽  
T D Fox
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Nuclear Gene ◽  
Small Subunit ◽  
Rrna Gene ◽  
Mitochondrial Translation ◽  
Activator Proteins ◽  
Translational Activator

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.

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