LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Wessels, Hans; Vogel, Rutger O.; Heuvel, Lambertus van den; Smeitink, Jan A.M.; Rodenburg, Richard J.; Nijtmans, Leo; Farhoud, Murtada H

doi:10.1002/pmic.200900157

Cited by 85 publications

(73 citation statements)

References 31 publications

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“…Of the two OXPHOS proteins that did increase during early lactation NDUFAF3 was the most notable since it displayed a 3.5-fold increase. Although this subunit of the OXPHOS pathway has only recently been described, it appears to play an important rate limiting role in the activity of complex 1 (55,72). Mutations in the gene encoding this protein have been linked to a fatal mitochondrial disease in human infants.…”

Section: Discussionmentioning

confidence: 99%

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

Hadsell

Olea

Wei

et al. 2011

Physiological Genomics

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Hadsell DL, Olea W, Wei J, Fiorotto ML, Matsunami RK, Engler DA, Collier RJ. Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle. Physiol Genomics 43: 271-285, 2011. First published December 28, 2010 doi:10.1152/physiolgenomics.00133.2010.-The regulation of mitochondrial biogenesis and function in the lactating mammary cell is poorly understood. The goal of this study was to use proteomics to relate temporal changes in mammary cell mitochondrial function during lactation to changes in the proteins that make up this organelle. The hypothesis tested was that changes in mammary cell mitochondrial biogenesis and function during lactation would be accounted for by coordinated changes in the proteins of the electron transport chain and that some of these proteins might be linked by their expression patterns to PPARGC1␣ and AMP kinase. The mitochondrial proteome was studied along with markers of mitochondrial biogenesis and function in mammary tissue collected from mice over the course of a single prolonged lactation cycle. Mammary tissue concentrations of AMP and ADP were increased (P Ͻ 0.05) during early lactation and then declined with prolonged lactation. Similar changes were also observed for mitochondrial ATP synthesis activity, mitochondrial mass and DNA copy number. Analysis of the mammary cell mitochondrial proteome identified 244 unique proteins. Of these, only two proteins of the electron transport chain were found to increase during early lactation. In contrast, coordinated changes in numerous electron transport chain proteins were observed both during mid-and late lactation. There were six proteins that could be directly linked to PPARGC1␣ through network analysis. Abundance of PPARGC-1␣ and phosphorylation of AMP kinase was highest on day 2 postpartum. The results suggest that the increases in mammary mitochondria ATP synthesis activity during early lactation results from changes in only a limited number proteins. In addition, decreases in a handful of proteins linked to lipid oxidation could be temporally linked to decreases in PPARGC1␣ and phospho-AMP kinase suggesting potential roles for these proteins in coordinating mammary gland metabolism during early lactation. mitochondria; proteome; adenosine 5=-triphosphate

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Section: Discussionmentioning

confidence: 99%

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

Hadsell

Olea

Wei

et al. 2011

Physiological Genomics

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“…Isolated mitochondria were solubilized with digitonin and homogenized, and the remaining membranes were removed by centrifugation. The supernatant was electrophoretically separated on a nondenaturating poly-acrylamide gel (37), individual bands were cut out and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (38,39), and peptides were annotated by Mascot (40). LacZ Constructs of Byps and Expression in E. coli.…”

Section: Methodsmentioning

confidence: 99%

Massive programmed translational jumping in mitochondria

Lang

Jakubkova

Hegedűsová

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Programmed translational bypassing is a process whereby ribosomes "ignore" a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a "takeoff codon" immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching "landing triplet" 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions.ribosome hopping | mitochondrial genome | proteome analysis | heterologous expression T he traditional view of translation is that mRNA is read sequentially, one codon at a time. However, low-level nonprogrammed translational bypassing (i.e., the occasional skipping of a few nucleotides) can be triggered by various factors, including tRNA paucity, unusual codons, and homo-polymer sequence tracts (1). In addition, programmed translational bypassing of 50 nt has been demonstrated for the gene 60 transcript of bacteriophage T4 (2-4). In vitro mutagenesis experiments showed that efficient translational "jumping" or "hopping" in T4 requires matching takeoff and landing codons (most effective is the wild-type GGA), a stop codon, and both a hairpin RNA secondary structure directly downstream of the takeoff site, and a Shine-Dalgarno (SD) sequence a few nucleotides upstream of the landing codon. Finally, a particular amino acid sequence in the nascent peptide encoded upstream of the takeoff site confers highest jumping efficiency. Additional cases of programmed bypassing have been postulated but currently lack supporting evidence (e.g., ref. 5), making the T4 gene 60 expression the only confirmed instance.Here, we report the massive occurrence of translational bypassing elements in mitochondria of the opportunistic human pathogen Magnusiomyces (also known as Blastoschizomyces or Geotrichum) capitatus (6), which belongs to a deeply branching lineage of Saccharomycetales (Fig. 1A). Our findings suggest that translational bypassing might be more widespread than previously thought. ResultsProtein-Coding Genes Interrupted by Dozens of In...

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“…Single-step affinity purification and nanospray ionization liquid chromatography tandem mass spectrometry and immunoblot analysis Single-step affinity purification and nano-LC-MS/MS and immunoblot analysis were performed as described previously (38). For single-step affinity purification of BOLA1-TAP and GLRX5-TAP, cells were resuspended in the lysis buffer provided with the InterPlay TAP Purification Kit (Agilent Technologies) and subjected to three cycles of freeze-thawing followed by 10-min centrifugation at 10,000 g. The supernatant was incubated for 2 h under rotation at 4°C in the presence of Strep-TactinSuperflow beads (IBA).…”

Section: Willems Et Almentioning

confidence: 99%

BOLA1 Is an Aerobic Protein That Prevents Mitochondrial Morphology Changes Induced by Glutathione Depletion

Willems

Wanschers

Esseling

et al. 2013

Antioxidants & Redox Signaling

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Aims: The BolA protein family is widespread among eukaryotes and bacteria. In Escherichia coli, BolA causes a spherical cell shape and is overexpressed during oxidative stress. Here we aim to elucidate the possible role of its human homolog BOLA1 in mitochondrial morphology and thiol redox potential regulation. Results: We show that BOLA1 is a mitochondrial protein that counterbalances the effect of L-buthionine-(S,R)-sulfoximine (BSO)-induced glutathione (GSH) depletion on the mitochondrial thiol redox potential. Furthermore, overexpression of BOLA1 nullifies the effect of BSO and S-nitrosocysteine on mitochondrial morphology. Conversely, knockdown of the BOLA1 gene increases the oxidation of mitochondrial thiol groups. Supporting a role of BOLA1 in controlling the mitochondrial thiol redox potential is that BOLA1 orthologs only occur in aerobic eukaryotes. A measured interaction of BOLA1 with the mitochondrial monothiol glutaredoxin GLRX5 provides hints for potential mechanisms behind BOLA1's effect on mitochondrial redox potential. Nevertheless, we have no direct evidence for a role of GLRX5 in BOLA1's function. Innovation: We implicate a new protein, BOLA1, in the regulation of the mitochondrial thiol redox potential. Conclusion: BOLA1 is an aerobic, mitochondrial protein that prevents mitochondrial morphology aberrations induced by GSH depletion and reduces the associated oxidative shift of the mitochondrial thiol redox potential. Antioxid. Redox Signal. 18, 129-138.

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LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Cited by 85 publications

References 31 publications

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

Developmental regulation of mitochondrial biogenesis and function in the mouse mammary gland during a prolonged lactation cycle

Massive programmed translational jumping in mitochondria

BOLA1 Is an Aerobic Protein That Prevents Mitochondrial Morphology Changes Induced by Glutathione Depletion

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