The Analysis of Yeast Mitochondrial Translation

Carlström, Andreas; Rzepka, Magdalena; Ott, Martin

doi:10.1007/978-1-0716-0834-0_17

Cited by 5 publications

(6 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Radiolabeling of mitochondrial translation products was done as described previously 62 . In brief, 2 OD 600 of cells were harvested after 6 h of growth on minimum media and incubated for 10 min at 30°C, shaking at 750 rpm.…”

Section: Methodsmentioning

confidence: 99%

Newly imported proteins in mitochondria are particularly sensitive to aggregation

et al. 2023

Self Cite

View full text Add to dashboard Cite

Aim A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation. Methods Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates. Results We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation. Conclusion Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress.

show abstract

Section: Methodsmentioning

confidence: 99%

Newly imported proteins in mitochondria are particularly sensitive to aggregation

et al. 2023

Self Cite

View full text Add to dashboard Cite

show abstract

“…[ 35 S]-methionine-based in vivo labeling of mitochondrial translation products was performed according to Carlström et al 35 with slight modifications. Cells were grown in SC medium containing galactose as carbon source (SC-Gal) to mid-logarithmic phase (approx.…”

Section: Methodsmentioning

confidence: 99%

“…To stop cytosolic translation, cycloheximide was added to a final concentration of 150 µg/mL and incubated for 2.5 min at 30°C, 600 rpm shaking. 3 µl of [ 35 S]-methionine (10 mCi/ml) were added to start the labeling reaction. For pulse-labeling, 200 µl aliquots were harvested after 5, 10 and 15 min, mixed with 50 µl of Stop solution (1.85 M NaOH; 1 M β-mercaptoethanol; 20 mM PMSF) and 10 µl of 200 mM cold methionine, and placed on ice.…”

Section: Methodsmentioning

confidence: 99%

“…Trichloroacetic acid was added to [ 35 S]-methionine-labeled samples with a final concentration of 14%, incubated for 30 min on ice and subsequently centrifuged for 30 min, 20.000 g at 4°C. Supernatants were carefully removed and pellets rinsed once in 1 ml 100% acetone.…”

Section: Methodsmentioning

confidence: 99%

“…30 µl of the sample were loaded on 16%/0.2% SDS polyacrylamide/bis-acrylamide gels. After separation, proteins were transferred to a nitrocellulose membrane, which was stained with Ponceau S. Protein standard bands (PageRuler TM Plus Prestained Protein Ladder, ThermoFisher) on the nitrocellulose membrane were marked with diluted [ 35 S]-methionine solution and the membranes were applied for autoradiography. Detection was performed with a Fujifilm FLA-9000 phosphorimager.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Multi-Omic Integration by Machine Learning (MIMaL) Reveals Protein-Metabolite Connections and New Gene Functions

Dickinson

Aufschnaiter

Ott

et al. 2022

Preprint

View full text Add to dashboard Cite

Cells respond to environments by regulating gene expression to exploit resources optimally. Recent advances in technologies allow the ability to gather information of cellular states of its components, measuring abundances of transcripts, their translation, the accumulation of proteins, lipids and metabolites. These highly complex datasets reflect the state of the different layers in a biological system. Multi-omics is the integration of these disparate methods and data to gain a clearer picture of the biological state. Multi-omic studies of the proteome and metabolome are becoming more common as mass spectrometry technology continues to be democratized. However, knowledge extraction through integration of these data remains challenging. Here we show that connections between omic layers can be discovered through a combination of machine learning and model interpretation. We find that model interpretation values connecting proteins to metabolites are valid experimentally and reveal also largely new connections. Further, clustering the magnitudes of protein control over all metabolites enabled prediction of gene five gene functions, each of which was validated experimentally. We accurately predicted that two uncharacterized genes in yeast modulate mitochondrial translation, YJR120W and YLD157C.We also predict and validate functions for several incompletely characterized genes, including SDH9, ISC1, and FMP52. Our work demonstrates that multi-omic analysis with machine learning (MIMaL) views multi-omic data through a new lens to reveal new insight that was not possible using existing methods.

show abstract