Whole cell formaldehyde cross-linking simplifies purification of mitochondrial nucleoids and associated proteins involved in mitochondrial gene expression.

Hodaka Fujii,Nina Rajala,Fenna Hensen,Daniel Ives,Hans J C T Wessels,Johannes N Spelbrink,Jolein Gloerich

doi:10.1371/journal.pone.0116726

Hodaka Fujii, Nina Rajala + Show 5 more

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https://doi.org/10.1371/journal.pone.0116726

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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.

Highlights

Mammalian mitochondrial DNA was discovered in the 1960 ́s[1,2] and early studies in 1969 by Nass suggested that mtDNA could be membrane bound[3]
Western blot analysis of these samples showed that several proteins implicated in mtDNA maintenance such as TFAM, POLG1 and Mitochondrial single stranded DNA binding protein (mtSSB) are enriched by FA crosslinking in TwinkleFLAG samples, following FLAG immuno affinity purification (IAP) (Fig. 1)
The results showed that following XL, TwinkleFLAG can be affinity-purified almost as efficiently as without XL and that in principle whole cell XL in combination with IAP can be used to enrich for nucleoid associated proteins

Summary

Introduction

Mammalian mitochondrial DNA (mtDNA) was discovered in the 1960 ́s[1,2] and early studies in 1969 by Nass suggested that mtDNA could be membrane bound[3]. The first microscopic observation of mtDNA as discrete structures within mitochondria came from the use of a DNA stain in the yeast S. cerevisiae. Despite this evidence and many additional studies in yeast and many other, often vertebrate, species (see below), mtDNA in mammals was for many years described as naked. This view has changed over the last 15–20 years and mtDNA is generally considered to be organized in discrete nucleo-protein complexes that are designated nucleoids by analogy to nucleo-protein complexes in bacteria[6,7]. TFAM and mtSSB were shown to colocalise with mtDNA in situ[14,15,16], the latter showing enrichment in particular with replicating nucleoids[17]

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