Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits

Schober, F.; Atanassov, Ilian; Moore, David; Calvo‐Garrido, Javier; Moedas, Marco F.; Wedell, Anna; Freyer, Christoph; Wredenberg, Anna

doi:10.1016/j.mcpro.2021.100065

Cited by 9 publications

(7 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Additonally, the impact of those phosphorylations on the localization of target proteins and, more specifically, the question of wheter mitochondrial proteins are phosphorylated before or after entering the mitochondria remain to be investigated. Accessibility, for instance, of previously undescribed phosphorylation sites on mitochondrial proteins can be assessed using advanced structural tools (Jumper et al ., 2021) to determine if they are likely targeted by a mitochondrial kinase or a cytoplasmic kinase before being imported (Schober et al, 2021). Moreover, future developments towards better enrichment strategies for the isolation of mitochondria from different tissues and advances in the MS technology will aid to further improve the depth and quality of the mitochondrial proteomes and phosphoproteome.…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial phosphoproteomes are functionally specialized across tissues

Hansen

Kremer

Karayel

et al. 2022

Preprint

View full text Add to dashboard Cite

Mitochondria are essential organelles involved in critical biological processes such as energy metabolism and cell survival. Their dysfunction is linked to numerous human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Furthermore, increasing evidence suggests that phosphorylation may also play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. We hypothesized that recent advances in mass spectrometry (MS)-based proteomics would now enable in-depth measurement to quantitatively profile mitochondrial proteomes along with their matching phosphoproteomes across tissues. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue modulated mitochondrial proteins (mitophos.de). We also uncover tissue-specific repertoires of dozens of kinases and phosphatases. Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on DRP1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.

show abstract

Section: Discussionmentioning

confidence: 99%

Mitochondrial phosphoproteomes are functionally specialized across tissues

Hansen

Kremer

Karayel

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Thus, any quantification must be thoroughly validated by orthogonal methods. Only stable isotope labeling by amino acids in cell culture (SILAC; Section 5.3.1 ) is likely to yield absolute quantification but is clearly not applicable to most sample types analyzed in proteomic studies (e.g., tissue and fluids); the exception may be Drosophila embryos fed labelled S. cerevisiae [ 186 , 187 ].…”

Section: Discovery Proteomicsmentioning

confidence: 99%

Proteomes Are of Proteoforms: Embracing the Complexity

2021

View full text Add to dashboard Cite

Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must assess the myriad of proteoforms that arise at any point before, after, and between translation and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as newly defined species. There are numerous analytical methods currently used to address proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. We must embrace the complexity. Yes, these are the hard questions, and this will not be easy…but where is the fun in easy?

show abstract

“…Finally, we performed immunoprecipitation (IP) experiments in combination with stable-isotope labelling of amino acids in fruit flies (SILAF) 63 to identify factors interacting with Dm ANGEL. With mass-spectrometry-based proteomics we identified, among others, enrichment for CG13850, a mitochondrial protein of unknown function (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Proteomics analysis was performed essentially as previously described 63 , 82 . Mouse heart tissue was homogenised in 8 M urea, 50 mM ammonium bicarbonate, and 400 μm LoBind silica beads followed by sonication.…”

Section: Methodsmentioning

confidence: 99%

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing

et al. 2022

Self Cite

View full text Add to dashboard Cite

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3′ phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3′ phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2.

show abstract

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits

Cited by 9 publications

References 62 publications

Mitochondrial phosphoproteomes are functionally specialized across tissues

Mitochondrial phosphoproteomes are functionally specialized across tissues

Proteomes Are of Proteoforms: Embracing the Complexity

ANGEL2 phosphatase activity is required for non-canonical mitochondrial RNA processing

Contact Info

Product

Resources

About