High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes

Sanglard, Lilian Vincis Pereira; Francs‐Small, Catherine Colas des

doi:10.1007/978-1-0716-1653-6_10

Cited by 7 publications

(8 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Another challenge was related to establishing high-throughput screening methods to link the phenotypes observed to mitochondrial defects. BN-PAGE of total membrane proteins followed by Western blotting [32] was used initially because this method uses the least amount of tissue to perform a rapid screen. Although this approach is not quantitative enough to detect minor changes in mitochondrial complexes and only covers some of the possible mitochondrial targets, one clear complex V mutant (transformant #280) was identified.…”

Section: Discussionmentioning

confidence: 99%

“…Most T2 transformants (177 out of 192) were screened through a simplified analysis of the mitochondrial respiratory complexes via blue native polyacrylamide gel electrophoresis (BN-PAGE) and Western blotting (WB). Considering the number of lines to screen and the small amount of tissue available in early generations, a crude membrane extraction protocol only requiring 100 mg of fresh tissue was used [12,32]. Wild types and the Nad6-deficient RPF2 variant (RPF2-nad6) mostly lacking assembled complex I [29] were used as controls on each gel.…”

Section: Analysis Of the Mitochondrial Respiratory Complexesmentioning

confidence: 99%

See 1 more Smart Citation

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Vincis Pereira Sanglard,

Small,

Colas des Francs-Small

2023

IJMS

View full text Add to dashboard Cite

Pentatricopeptide repeat (PPR) proteins are considered a potential tool for manipulating organelle gene expression in plants because they can recognise a wide range of different RNA sequences, and the molecular basis for this sequence recognition is partially known and understood. A library of redesigned PPR proteins related to restorer-of-fertility proteins was created and transformed into plants in order to target mitochondrial transcripts. Ninety different variants tested in vivo showed a wide range of phenotypes. One of these lines, which displayed slow growth and downward curled leaves, showed a clear reduction in complex V. The phenotype was due to a specific cleavage of atp1 transcripts induced by a modified PPR protein from the library, validating the use of this library as a source of mitochondrial ‘mutants’. This study is a step towards developing specific RNA targeting tools using PPR proteins that can be aimed at desired targets.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Analysis Of the Mitochondrial Respiratory Complexesmentioning

confidence: 99%

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Vincis Pereira Sanglard,

Small,

Colas des Francs-Small

2023

IJMS

View full text Add to dashboard Cite

show abstract

“…BN-PAGE, SDS-PAGE and western blotting were performed as previously described (Colas des Francs-Small et al, 2014; Vincis Pereira Sanglard and Colas des Francs-Small, 2022). The antibodies used in this work are listed in Supplemental Table 10.…”

Section: Methodsmentioning

confidence: 99%

Knockdown of mitochondrialatp1mRNA by a custom-designed pentatricopeptide repeat protein alters F₁F_oATP synthase

Yang

Sanglard

Lee

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

We show that a custom-designed RNA-binding protein binds and specifically induces cleavage of atp1 RNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis thaliana. The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits, lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP-synthase-depleted lines showed higher respiratory rates and elevated levels of most amino acids at night, most notably serine family amino acids. The results show the value of using custom-designed PPR proteins to influence expression of specific mitochondrial transcripts to carry out reverse genetics studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis.

show abstract

“…A panel of several state-of-the-art analytical methods is often used to identify possible stable protein complexes. These include yeast two-hybrid assay [ 10 ], the chemical cross-linking approach [ 11 , 12 ], density-gradient ultracentrifugation [ 13 ], co-immunoprecipitation, and tandem affinity purification [ 14 , 15 , 16 ], as well as blue native gel electrophoresis [ 17 , 18 ].…”

Section: Interactome Profiling Of Stable Protein Complexesmentioning

confidence: 99%

Protein Interactome Profiling of Stable Molecular Complexes in Biomaterial Lysate

Mezentsev

Ершов

Yablokov

et al. 2022

IJMS

View full text Add to dashboard Cite

Most proteins function as part of various complexes, forming via stable and dynamic protein–protein interactions (PPIs). The profiling of PPIs expands the fundamental knowledge about the structures, functions, and regulation patterns of protein complexes and intracellular molecular machineries. Protein interactomics aims at solving three main tasks: (1) identification of protein partners and parts of complex intracellular structures; (2) analysis of PPIs parameters (affinity, molecular-recognition specificity, kinetic rate constants, and thermodynamic-parameters determination); (3) the study of the functional role of novel PPIs. The purpose of this work is to update the current state and prospects of multi-omics approaches to profiling of proteins involved in the formation of stable complexes. Methodological paradigm includes a development of protein-extraction and -separation techniques from tissues or cellular lysates and subsequent identification of proteins using mass-spectrometry analysis. In addition, some aspects of authors’ experimental platforms, based on high-performance size-exclusion chromatography, procedures of molecular fishing, and protein identification, as well as the possibilities of interactomic taxonomy of each protein, are discussed.

show abstract

High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes

Cited by 7 publications

References 15 publications

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Knockdown of mitochondrialatp1mRNA by a custom-designed pentatricopeptide repeat protein alters F₁F_oATP synthase

Protein Interactome Profiling of Stable Molecular Complexes in Biomaterial Lysate

Contact Info

Product

Resources

About

High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes

Cited by 7 publications

References 15 publications

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Alteration of Mitochondrial Transcript Expression in Arabidopsis thaliana Using a Custom-Made Library of Pentatricopeptide Repeat Proteins

Knockdown of mitochondrialatp1mRNA by a custom-designed pentatricopeptide repeat protein alters F1FoATP synthase

Protein Interactome Profiling of Stable Molecular Complexes in Biomaterial Lysate

Contact Info

Product

Resources

About

Knockdown of mitochondrialatp1mRNA by a custom-designed pentatricopeptide repeat protein alters F₁F_oATP synthase