Monitoring mitochondrial translation by pulse SILAC

Imami, Koshi; Selbach, Matthias; Ishihama, Yasushi

doi:10.1101/2021.01.31.428997

Cited by 5 publications

(5 citation statements)

References 65 publications

(116 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For mass spectrometry analysis, beads were washed twice using buffer D (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 15 mM MgCl2) after washing with buffer B. We followed the original protocol (Imami et al 2018(Imami et al , 2023 with some modifications. Beads were resuspended in PTS buffer (12 mM sodium deoxycholate (SDC), 12 mM sodium Nlauroylsarcosinate (SLS) in 100 mM Tris-HCl pH 8.0) followed by incubation for 30 min at 37°C under constant agitation.…”

Section: Immunoprecipitationmentioning

confidence: 99%

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

Ugajin

Imami

Takada

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.

show abstract

Section: Immunoprecipitationmentioning

confidence: 99%

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

Ugajin

Imami

Takada

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Therefore, mitochondrial translation has been researched by several methods [see Apostolopoulos and Iwasaki (2022) for a summary], such as 35 S-methionine-labeling of newly synthesized nascent chains ( Chomyn, 1996 ; Sasarman and Shoubridge, 2012 ), the quantification of ribosome-associated mRNAs by quantitative reverse transcription-PCR ( Antonicka et al, 2013 ; Fung et al, 2013 ; Zhang et al, 2014 ; Pearce et al, 2017 ), ribosome profiling ( Rooijers et al, 2013 ; Iwasaki et al, 2016 ; Gao et al, 2017 ; Pearce et al, 2017 ; Morscher et al, 2018 ; Suzuki et al, 2020 ; Kashiwagi et al, 2021 ; Li et al, 2021 ; Schöller et al, 2021 ), and pulse stable isotope labeling by amino acids in cell culture for proteomic analysis ( Imami et al, 2021 ). However, these approaches provide averaged data for thousands of cells, which are pooled for the experiments, and thus pose an analytic hurdle when assessing mitochondrial translation in each individual cell.…”

Section: Introductionmentioning

confidence: 99%

High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS

Saito¹,

Osaki²,

Ikeuchi³

et al. 2023

BIO-PROTOCOL

View full text Add to dashboard Cite

In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

show abstract

“…Owing to the recent emergence of structures at near-atomic resolutions, the architecture of mitoribosomes has been revealed to be distinct from bacterial and eukaryotic cytoplasmic ribosomes (or cytoribosomes) ( Kummer and Ban 2021 ). In organello translation has been investigated through various approaches, such as the following: (1) sucrose density gradient ultracentrifugation for polysome isolation and subsequent mRNA quantification via quantitative reverse transcription-PCR (RT-qPCR) or northern blotting ( Antonicka et al 2013 ; Fung et al 2013 ; Grimes et al 2014 ; Zhang et al 2014 ; Pearce et al 2017 ; Cahoon and Qureshi 2018 ); (2) ribosome profiling ( Iwasaki et al 2016 ; Suzuki et al 2020 ; Kashiwagi et al 2021 ), a method based on RNase footprinting by ribosomes and following deep sequencing ( Ingolia et al 2009 ), and the derivative methods tailored for mitochondrial translation ( Rooijers et al 2013 ; Couvillion et al 2016 ; Couvillion and Churchman 2017 ; Gao et al 2017 ; Pearce et al 2017 ; Morscher et al 2018 ; Li et al 2021 ; Schöller et al 2021 ); (3) pulsed stable isotope labeling by amino acids in cell culture (pSILAC) following mass spectrometry ( Imami et al 2021 ); (4) in situ cryo-electron tomography ( Pfeffer et al 2015 ; Englmeier et al 2017 ); (5) in vitro reconstitution with purified factors ( Lee et al 2021 ); and (6) in vitro silencing of translation in purified mitochondria ( Cruz-Zaragoza et al 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Kimura

Saito

Osaki

et al. 2022

RNA

View full text Add to dashboard Cite

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

show abstract

Monitoring mitochondrial translation by pulse SILAC

Cited by 5 publications

References 65 publications

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development

High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation

Contact Info

Product

Resources

About