Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

Abstract

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

Figure 1.

Fractionation of mitochondrial nucleoprotein complexes on iodixanol gradients. (A) Mitochondrial 1000g_max supernatants from HEK293T cells were fractionated on 20–42.5% iodixanol gradients, and extracted nucleic acids from each fraction were separated by agarose gel electrophoresis and stained with ethidium bromide. (B) Protease-treated mitochondrial nucleic acids fractionated on a 20–42.5% iodixanol gradient. (C) The chart shows the relative abundance of five mitochondrial RNAs (12S rRNA, 16S rRNA, cox2, cytb and nd1) based on quantitative RT-PCR (see ‘Materials and Methods’ section); above the chart is a transilluminator image of the ethidium bromide-stained RNA from each fraction. The qPCR results for the three mRNAs (cox2, cytb and nd1) were similar and so they were grouped together (blue line). Each fraction was ‘spiked’ with an equal amount of synthetic GFP transcript (green line), as a control. Quantitative RT-PCR of 18S rRNA was performed in parallel, but none was detected, indicating that any contamination with RNA of cytosolic ribosomes was below the limits of detection. (D) Ethidium bromide staining and qPCR detection of mtDNA based on the amplification of five mitochondrial genes (see ‘Materials and Methods’ section). (E) The DNA and RNA detected by transillumination from panels (C) and (D) are displayed above a series of immunoblots for a range of mitochondrial proteins (see text for details). The boxed area includes all the results for fraction 8, in which mRNA peaked. (F) Immunoblots of mitochondrial 1000g_max supernatants of HEK cells before (input) and after IP with anti-ATAD3 antibody. The control was instead incubated with preimmune serum. (G) The products of mitochondrial protein synthesis were labelled with ³⁵S-methionine in 143B cells subjected to one or two rounds of transfection with dsRNA1 targeting ATAD3 (R_i) or mock transfected (C) cells. Large gel image: mitochondrial translation products at the base of the panel are immunoblots of the same cell lysates probed with antibodies to ATAD3 or GAPDH.

Figure 2.

Elevated expression of HA tagged ATAD3B impedes mtDNA replication by concentrating the protein in foci separate from mitochondrial nucleoids. (A) HEK293T cells carrying an ATAD3B transgene with an HA tag were induced to express the recombinant protein with 50 ng/ml doxycyline for 6 days. DNA was harvested daily and the mtDNA copy number assayed by qPCR. Controls were HEK293T cells transformed with an empty vector and maintained in the same dose of drug. (B) qPCR estimation of mtDNA copy number after harvesting DNA from cells expressing ATAD3B.HA protein, or control cells, at daily intervals, having first reduced mtDNA copy number to 20% of normal by exposure to 50 ng/ml ethidium bromide for 72 h. The cells were maintained in 50 ng/ml doxycycline throughout the experiment. For charts (A) and (B), n = 3 experiments, error bars are 1 SD from the mean. The distribution of mitochondrial nucleoids (anti-DNA antibody) and ATAD3B.HA (anti-HA antibody) was determined by confocal microscopy 24, 72 and 120 h after induction of the transgene with 50 ng/ml doxycycline [panels (C–E), respectively].

Figure 3.

Elevated expression of ATAD3B.HA perturbs mtDNA topology, protein-dependent mtDNA multimers and mitochondrial replication intermediates. (A) HEK293T cells expressing ATAD3B.HA for 5 days were DNA stained with picogreen and the mitochondrial network was labelled with mitotracker orange (pseudo-coloured red for contrast). (B) Mitochondrial nucleoprotein was isolated from HEK293T cells expressing ATAD3B.HA for 3 or 5 days (50 ng/ml doxycycline), and from (control) cells cultured without the drug. The DNA was digested with AccI, separated by 2D-AGE and hybridized to a probe detecting the major non-coding region of human mtDNA [see Ref. (41) for a detailed exposition of the protein-dependent multimers of mtDNA labelled x₂, x₃, d and w]. (C) Mitochondrial replication intermediates of cells induced to express ATAD3B.HA and controls. Samples and processing were identical to panel B except that the DNA was treated additionally with proteinase K during the isolation procedure. Note the enhanced standard replication fork (y) arc after 120 h expression of ATAD3B.HA in panels (B) and (C).

Figure 4.

Decreased expression of prohibitin impairs mitochondrial protein synthesis. (A) Mitochondrial translation was assayed in mock transfected 143B cells (C) or cells transfected with a scrambled dsRNA (Sc), or with dsRNA targeting PHB1 (R_i) and separated by SDS–PAGE (4–12% Nu-PAG, Invitrogen). Decreased expression of the target protein was confirmed by immunoblotting of PHB1 with GAPDH as the reference protein. (B) The PHB2 gene was ablated in 90% of cells by incubating MEFs, whose sole copy of PHB2 is flanked by flox sites, with cre-recombinase [Supplementary Figure S10-A and Ref. (42)]. C, control MEFs, PHB^flx/flx MEFs with flox sites flanking the prohibitin 2 (PHB2) gene. Cre, cre-recombinase that excises the PHB2 gene; −, no cre added; +, one dose of cre; ++, two doses of cre (see Supplementary Figure S10-A).

Figure 5.

CRIF1, with C-terminal FLAG-StrepII tags, is targeted exclusively to mitochondria and gene-silencing of CRIF1 impairs mitochondrial protein synthesis. (A) After transient transfection of HOS cells with a CRIF1.FLAG.StepII cDNA cloned in pcDNA5 (Invitrogen), the cells were labelled with DAPI, mitotracker orange (pseudo-coloured red for contrast) and anti-CRIF1 (green). (B) Mitochondrial translation products in mock transfected HOS cells (C) or cells transfected with 10 nM dsRNA, G₁ or G₅ targeting CRIF1 (R_i), as per ATAD3 (see Figure 1g and ‘Materials and Methods’ section).