Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

Abstract

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.

Figure 1. The majority of the cholesterol in the mitochondrial inner membrane co-fractionates with the membrane associated nucleoid.

Pellet (a) and supernatant (b) preparations from digitonin treated purified mitochondria from HEK293 cells were separated on bottom-up flotation iodixanol gradients and their protein, DNA and lipid contents were analysed. The mtDNA and associated nucleoid proteins (Twinkle, polG, TFAM, SSB) as well as most of the cholesterol migrate in the same fraction in the pellet gradient. Some of the MAM marker protein FACL4 co-fractionates with the nucleoid in fraction 8. mtDNA, mitochondrial DNA; polG, polymerase Gamma; SSB, single-stranded binding protein; TFAM, mitochondrial transcription factor A; PHB, prohibitin, MRPS, mitochondrial ribosomal protein small subunit; MRPL, mitochondrial ribosomal protein large subunit; Clnx, calnexin; coxII, cytochrome oxidase subunit II; TOM20, translocase of outer membrane 20 kDa subunit; VDAC1, voltage-dependent anion channel 1. The lipid data is from the analyses of three replicate gradients (SEM), the protein and DNA profiles are from one representative gradient, while all three gradients were tested with at least the Twinkle, mtSSB, coxII, TFAM and ATAD3 antibodies by Western blot analysis and dot-blot analysis for mtDNA. The antibody probings were done on multiple blots (typically 2–3) with the same fractions to allow for the multiple detections with the many antibodies used.

Figure 2. Nucleoid components and cholesterol co-sediment in the absence of mitochondrial DNA.

Total mitochondrial lauryl-maltoside lysates from mtDNA containing control cells (a) and ρ° cells (b) were separated on top-down iodixanol gradients. Though some of the nucleoid component are lost or have moved to the top of the gradient in the absence of mtDNA, some still co-sediment at the bottom of the gradient. Pellet preparations from digitonin treated purified mitochondria from ρ° cells were separated on a bottom-up flotation iodixanol gradient (c). The nucleoid-protein and cholesterol fractions remain intact and co-fractionate also in the absence of mtDNA. mtDNA, mitochondrial DNA; TFAM, mitochondrial transcription factor A; SSB, single-stranded binding protein; polG, polymerase Gamma; POLRMT, mitochondrial RNA polymerase; HSP60, heat shock protein 60; coxII, cytochrome oxidase subunit II, PHB, prohibitin; Cyp-D, Cyclophilin D. Protein, DNA and lipid profiles of representative gradients.

Figure 3. Knock-down of ATAD3 disrupts the cholesterol containing fraction and with it the membrane associated nucleoid.

Pellet preparations from digitonin treated purified mitochondria derived from ATAD3 siRNA treated HEK293 cells were separated on a bottom-up flotation iodixanol gradient. The cholesterol and corresponding nucleoid components no longer migrate tightly as one fraction, but are spread evenly over two fractions. mtDNA, mitochondrial DNA; polG, polymerase Gamma; SSB, single-stranded binding protein; TFAM, mitochondrial transcription factor A; PHB, prohibitin, MRPS, mitochondrial ribosomal protein small subunit; MRPL, mitochondrial ribosomal protein large subunit; Clnx, calnexin; coxII, cytochrome oxidase subunit II; TOM20, translocase of outer membrane 20 kDa subunit; VDAC1, voltage-dependent anion channel 1. Protein, DNA and lipid profiles of replicate gradients are presented in SFig. 2.

Figure 4. Nucleoid components are detected in purified MAM.

Sub-cellular fractionations of HEK293 cells were done according to Western-blot analyses using different markers to identify cytosolic (actin), ER (CALNX), mitochondrial (coxI, coxII, Twinkle, TFAM, cyclophilin D, ATAD3) and MAM (FACL4) components showed that in HEK293 cells, mitochondria cannot be easily separated from ER membranes. However, some MAM can be freed from mitochondrial membranes as evidenced by the absence of the mitochondrial membrane markers coxI and coxII. Mitochondrial nucleoid components are still detected in this otherwise mitochondria-free MAM-preparation (a). The amount of cholesterol (molar percentage of total lipids) in MAM is higher than in purified mitochondria derived from HEK293 cells (mt N =3 ; MAM N = 3) (b). ER, endoplasmic reticulum; MT, mitochondria; MAM, mitochondria associated membranes; FACL4, long-chain acyl-CoA synthetase 4; CALNX, calnexin; coxI cytochrome oxidase subunit I; coxII, cytochrome oxidase subunit II; CyP-d, Cyclophilin D; TFAM, mitochondrial transcription factor A.

Figure 5. Knock-down of ATAD3 affects mitochondrial membrane structure.

TEM images of cells transfected with a scrambled siRNA construct (b) and a siRNA against ATAD3 (a) reveal changes in the mitochondrial membranes, including abnormal cristae, in the absence of ATAD3 compared with the mitochondria of mock transfected cells.

Figure 6. The cholesterol-rich fraction and the associated nucleoid remain intact after the knock-down of mitofilin.

Pellet preparations from digitonin treated purified mitochondria derived from mitofilin siRNA treated HEK293 cells were separated on a bottom-up flotation iodixanol gradient (a). The majority of the cholesterol and corresponding nucleoid components migrate to the same fraction as in control preparations (Fig. 1). Mitofilin knock-down (b). TFAM, mitochondrial transcription factor A; SSB, single-stranded binding protein; coxII, cytochrome oxidase subunit II. Protein, DNA and lipid profiles of a representative gradient.