Whole Cell Formaldehyde Cross-Linking Simplifies Purification of Mitochondrial Nucleoids and Associated Proteins Involved in Mitochondrial Gene Expression

Abstract

Mitochondrial DNA/protein complexes (nucleoids) appear as discrete entities inside the mitochondrial network when observed by live-cell imaging and immunofluorescence. This somewhat trivial observation in recent years has spurred research towards isolation of these complexes and the identification of nucleoid-associated proteins. Here we show that whole cell formaldehyde crosslinking combined with affinity purification and tandem mass-spectrometry provides a simple and reproducible method to identify potential nucleoid associated proteins. The method avoids spurious mitochondrial isolation and subsequent multifarious nucleoid enrichment protocols and can be implemented to allow for label-free quantification (LFQ) by mass-spectrometry. Using expression of a Flag-tagged Twinkle helicase and appropriate controls we show that this method identifies many previously identified nucleoid associated proteins. Using LFQ to compare HEK293 cells with and without mtDNA, but both expressing Twinkle-FLAG, identifies many proteins that are reduced or absent in the absence of mtDNA. This set not only includes established mtDNA maintenance proteins but also many proteins involved in mitochondrial RNA metabolism and translation and therefore represents what can be considered an mtDNA gene expression proteome. Our data provides a very valuable resource for both basic mitochondrial researchers as well as clinical geneticists working to identify novel disease genes on the basis of exome sequence data.

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

“…All 37 of our proteins are known mitochondrial proteins, not cytosolic or nuclear contaminants, which have been observed in all previous nucleoid MS studies (Figure S1A). To determine nucleoid specificity, we checked for nucleoid-related annotation in the literature, or detection in three of the highest-quality previous nucleoid MS datasets (Bogenhagen et al, 2008; Rajala et al, 2015; Wang and Bogenhagen, 2006). Thirty proteins, or 81% of our list, have a prior nucleoid link by these criteria (Figure 2E).…”

“…The nucleoid is not surrounded by a membrane, and is thought to be a dynamic structure with transiently associated components (Gilkerson et al, 2013). Previous efforts to map the nucleoid by mass spectrometry (MS) have used either crosslinking followed by fractionation (Bogenhagen et al, 2008; Kaufman et al, 2000; Rajala et al, 2015) or immunoprecipitation of known nucleoid proteins (He et al, 2012; Rajala et al, 2015; Wang and Bogenhagen, 2006). The former approach retrieves contaminants, including even cytosolic and nuclear proteins (Bogenhagen et al, 2008; Rajala et al, 2015) (Figure S1A), while the latter misses transient interaction partners that do not survive cell lysis and detergent washes.…”

Organic acid production in Aspergillus niger and other filamentous fungi

“…LC-MS/MS analysis was performed at Radboud Proteomics Centre as described previously (Rajala et al, 2015). Measurements were performed by nanoflow reversed-phase C18 liquid chromatography (EASY nLC, Thermo Scientific) coupled online to a 7 Tesla linear ion trap Fourier-Transform ion cyclotron resonance mass spectrometer (LTQ FT Ultra, Thermo Scientific).…”

“…analyses were done at Radboud Proteomics Centre as described previously (Rajala et al, 2015). The obtained raw mass spectrometry proteomics data was deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD004909.…”

The uptake of carbon sources by Aspergillus niger

“…The protocol used in order to prepare membrane proteins for LC-MS/MS analysis has been described in detail previously [36]. LC-MS/MS analyzes were done at Radboud Proteomics Centre as described previously [74]. The obtained raw mass spectrometry proteomics data was deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD004909.…”