scholarly journals Monitoring mitochondrial translation by pulse SILAC

2021 ◽  
Author(s):  
Koshi Imami ◽  
Matthias Selbach ◽  
Yasushi Ishihama
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Membrane Proteins ◽  
Translational Regulation ◽  
Isotope Labeling ◽  
Protein Complexes ◽  
Complex Iii ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Hydrophobic Nature

SummaryMitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.

scholarly journals Mass spectrometry of membrane protein complexes

2019 ◽  
Vol 400 (7) ◽  
pp. 813-829 ◽  
Author(s):  
Julian Bender ◽  
Carla Schmidt
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Three Dimensional ◽  
Membrane Protein Complexes ◽  
Hydrophobic Nature ◽  
Alternative Approaches ◽  
Lipid Environment ◽  
And Function

Abstract Membrane proteins are key players in the cell. Due to their hydrophobic nature they require solubilising agents such as detergents or membrane mimetics during purification and, consequently, are challenging targets in structural biology. In addition, their natural lipid environment is crucial for their structure and function further hampering their analysis. Alternative approaches are therefore required when the analysis by conventional techniques proves difficult. In this review, we highlight the broad application of mass spectrometry (MS) for the characterisation of membrane proteins and their interactions with lipids. We show that MS unambiguously identifies the protein and lipid components of membrane protein complexes, unravels their three-dimensional arrangements and further provides clues of protein-lipid interactions.

scholarly journals C-terminal tail length guides insertion and assembly of membrane proteins

2020 ◽  
Vol 295 (46) ◽  
pp. 15498-15510 ◽  
Author(s):  
Sha Sun ◽  
Malaiyalam Mariappan
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Molecular Chaperones ◽  
Time Window ◽  
Protein Complexes ◽  
Tail Length ◽  
Protein Components ◽  
Partner Proteins ◽  
Erad Pathway

A large number of newly synthesized membrane proteins in the endoplasmic reticulum (ER) are assembled into multiprotein complexes, but little is known about the mechanisms required for assembly membrane proteins. It has been suggested that membrane chaperones might exist, akin to the molecular chaperones that stabilize and direct the assembly of soluble protein complexes, but the mechanisms by which these proteins would bring together membrane protein components is unclear. Here, we have identified that the tail length of the C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. We found that membrane proteins with C-TMD tails shorter than ∼60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion. These C-TMDs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMDs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD) pathway for removal. In contrast, C-TMDs with sufficient hydrophobicity or tails longer than ∼80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, our data suggest that C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins.

scholarly journals Cardiolipin is required for membrane docking of mitochondrial ribosomes and protein synthesis

2020 ◽  
Vol 133 (14) ◽  
pp. jcs240374 ◽  
Author(s):  
Richard G. Lee ◽  
Junjie Gao ◽  
Stefan J. Siira ◽  
Anne-Marie Shearwood ◽  
Judith A. Ermer ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Stress Responses ◽  
Protein Complexes ◽  
Mitochondrial Protein ◽  
Mitochondrial Morphology ◽  
Mitochondrial Translation ◽  
Inner Membrane ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Ribosome

ABSTRACTThe mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the CL biosynthesis gene Crls1 to investigate the effects of CL loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by uncoordinated mitochondrial translation rates and impaired respiratory chain supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of CL resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that CL is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 (also known as OXA1L) during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.

Antioxidant and Enzyme Inhibitory Properties of Mangifera indica leaf Extract

2020 ◽  
Vol 10 (4) ◽  
pp. 384-394
Author(s):  
Sainiara Begum ◽  
Archana Banerjee ◽  
Bratati De
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Leaf Extract ◽  
Methanol Extract ◽  
Mangifera Indica ◽  
Agricultural Waste ◽  
Ethyl Acetate Fraction ◽  
Soil Amelioration ◽  
Liver Disorders ◽  
Inhibitory Activities

Aims:The foliar residues of Mangiferaindica tree are usually burned or used for soil amelioration except nominal uses as fodder.Methods:To add value to this agricultural waste, extracts of the leafy residues of M. indica were studied to analyze their potential as antioxidants and to inhibit the enzymes related to the management of diabetes, Alzheimer’s Disease (AD), hepatic disorders as well as to identify important phytochemicals present in the extracts.Results:Results depicts that the leaves have notable bioactivities. The methanol extract (ME) showed much potential than ethyl acetate fraction after hydrolysis (HME) against α-amylase and α- glucosidase. The activity against the enzyme β-glucuronidase was also higher than that of the commercial β- glucuronidase inhibitor. The extract after hydrolysis showed better antioxidant and acetylcholinesterase inhibitory activities. Detection of important phytochemicals such as chrysin and myricetn, alizarin, arbutin, hydroquinone, tyrosol, taxifolin, kaempferol, mangiferin, and the vitamin alpha tocophereol, in addition to a number of organic acids, amino acids, fatty acids, sugars and polyols by GC-MS and HPTLC based analysis of the extract of M. indica leaf, also suggest the use of the leaves as sources of these important phytochemicals.Conclusion:More concisely HME with more number of detected metabolites found better to be used against oxidative stress as well as enzymes related to neural and liver disorders than that of ME.

scholarly journals Amino Acid and Acylcarnitine Levels in Chronic Patients with Schizophrenia: A Preliminary Study

Metabolites ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 34
Author(s):  
Irina A. Mednova ◽  
Alexander A. Chernonosov ◽  
Marat F. Kasakin ◽  
Elena G. Kornetova ◽  
Arkadiy V. Semke ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Amino Acid ◽  
Chronic Schizophrenia ◽  
Integrated Approach ◽  
Serum Levels ◽  
Tandem Mass ◽  
Chronic Patients ◽  
Healthy Donors ◽  
Preliminary Study

Amino acids and acylcarnitines play an important role as substrates and intermediate products in most of pathways involved in schizophrenia development such as mitochondrial dysfunction, inflammation, lipid oxidation, DNA damage, oxidative stress, and apoptosis. It seems relevant to use an integrated approach with ‘omics’ technology to study their contribution. The aim of our study was to investigate serum amino acid and acylcarnitine levels in antipsychotics-treated patients with chronic schizophrenia compared with healthy donors. We measured serum levels of 15 amino acids and 30 acylcarnitines in 37 patients with schizophrenia and 36 healthy donors by means of tandem mass spectrometry. In summary, patients with chronic schizophrenia had an altered concentration of a few amino acids and acylcarnitines in comparison to the healthy probands. Further research is needed to assess and understand the identified changes.

Can keto/amino acids reduce oxidative stress in peritoneal dialysis patients with hypoalbuminemia?

2021 ◽  
Author(s):  
Erdem Çankaya ◽  
Yusuf Bilen ◽  
Abdullah Uyanık ◽  
Hasan Dogan ◽  
Ahmet Kızıltunç ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Amino Acids ◽  
Peritoneal Dialysis ◽  
Dialysis Patients

scholarly journals Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane

Life ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 15
Author(s):  
Radek Kaňa ◽  
Gábor Steinbach ◽  
Roman Sobotka ◽  
György Vámosi ◽  
Josef Komenda
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Protein Interactions ◽  
Thylakoid Membrane ◽  
Protein Complexes ◽  
Correlation Spectroscopy ◽  
Membrane Microdomains ◽  
Fast Diffusion ◽  
Light Harvesting Complexes ◽  
Term Survival

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

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