Initiation Factor 3 is Dispensable For Mitochondrial Translation in Cultured Human Cells

Abstract

The initiation of protein synthesis in bacteria is ruled by three canonical factors: IF1, IF2, and IF3. This system persists in human mitochondria; however, it functions in a rather different way due to specialization and adaptation to the organellar micro-environment. We focused on human mitochondrial IF3, which was earlier studied in vitro, but no knock-out cellular models have been published up to date. In this work, we generated human HeLa cell lines deficient in the MTIF3 gene and analyzed their mitochondrial function. Despite the lack of IF3mt in these cells, they preserved functional mitochondria capable of oxygen consumption and protein synthesis; however, the translation of ATP6 mRNA was selectively decreased which compromised the assembly of ATP synthase. Together with the analogous results obtained earlier for baker's yeast mitochondrial IF3, our findings point to a functional divergence of mitochondrial initiation factors from their bacterial ancestors.

Figure 1

Disruption of MTIF3 gene in HeLa cells. (A) The structure of the MTIF3 gene is represented according to the Ensembl genome browser. Three alternatively spliced mRNAs (red) and one protein (blue) are shown. Transcripts are drawn as boxes (exons) and lines connecting the boxes (introns). Filled boxes represent coding sequence and unfilled boxes (or portions of boxes) represent UnTranslated Regions (UTR). For coding transcripts (red), protein motifs and domains are shown in blue. Two crossing parallel lines indicate the long genome regions of the introns which were not shown to keep the scale of the scheme. (B) The results of the sequencing of the DNA of the mutant cells. The coincident DNA sequences are in brown boxes, coincident protein sequences are in blue boxes. Protein sequences typed with red letters are the mitochondrial transit peptide (MTS). The sequences start from the first ATG codon and first methionine respectively. The region of the mutant gene where the deletion takes place is indicated by an arrow. Only partial sequences demonstrating the frame-shift are shown. (C) The results of the IF3mt immunodetection by Western blot (left panel). WT: wild type cells; MTIF3 KO: mutant cells with a disrupted MTIF3 gene; 1 and 2 are separate clones. The IF3mt band is marked by an arrow. The positions of the protein molecular weight markers together with their weights in kDa are depicted on the left. As a loading control, the gel with the same samples loaded was stained by Coomassie R250 (right panel). (D) RT-qPCR analysis of MTIF3 gene expression. WT: wild type cells; MTIF3 KO: mutant cells with disrupted MTIF3 gene. GAPDH gene expression serves as an internal control. The data were calculated following the ((Ct algorithm and normalized on the signal of the β-actin mRNA primer pair. The error bars were built based on three independent experiments.

Figure 2

Analysis of the mitochondrial function of MTIF3 knock-out cells. The designations are the same for all panels (WT: wild type cells; MTIF3 KO: mutant cells with disrupted MTIF3 gene; 1 and 2 are separate clones). (A) Growth curves of WT and MTIF3 KO cells in glucose- or galactose-containing media. The error bars were built based on three independent experiments. (B) Analysis of the cell viability on glucose- or galactose-containing media in the indicated periods of time. The error bars were built based on three independent experiments. (C) The radioautograph of labeled mitochondrial translation products separated by electrophoresis in 15–20% gradient PAAG (left panel). Cell lines used for the experiment are depicted on the top. Individual mitochondrial proteins are marked on the left. The asterisk marks the band of ATP6. As loading control, the gel was stained with Coomassie R250 (right panel). The experiment was done three times; the typical picture is shown. (D) Calculations of the relative quantities of mitochondrial translation products shown in (C). The frame marks the reduction of ATP6 amount as a result of MTIF3 gene disruption. The error bars were built based on three independent experiments. (E) The staining of complex V activity. Cell lines used for the experiment are depicted on the top. The positions of complete F0F1 enzyme and free F1 subunit are indicated. (F) The staining of complex I activity. Cell lines used for the experiment are depicted on the top. SC: supercomplexes, CI: free complex I. The weak bands below free complex I are the residual signals of the complex V staining. (G) Oxygen consumption in the wild type HeLa and MTIF3 mutant cells. Quantification of coupled and uncoupled respiration is presented (indicated on the bottom). The error bars were built based on four independent experiments.

Figure 3

Analysis of the mitochondrial function of MTIF3 knock-out cells rescued by the MTIF3-coding plasmid. The designations are the same for all panels (WT + vector: wild type cells transfected with the empty vector; MTIF3 KO + vector: mutant cells with disrupted MTIF3 gene transfected with the empty vector; WT rescued: wild type cells transfected with the MTIF3-coding plasmid; MTIF3 KO rescued: mutant cells with disrupted MTIF3 gene transfected with the MTIF3-coding plasmid). (A) The results of the IF3mt immunodetection by Western blot (left panel). Cell lines used for the experiment are depicted on the top. The bands of the native IF3mt and the fusion protein IF3mt-HA are marked with the arrows. The positions of the protein molecular weight markers together with their weights in kDa are depicted on the left. As a loading control, the gel with the same samples loaded was stained by Coomassie R250 (right panel). (B) The radioautograph of labeled mitochondrial translation products separated in 15–20% gradient PAAG (left panel). Cell lines used for the experiment are depicted on the top. Individual mitochondrial proteins are marked on the left. The asterisk marks the band of ATP6. As loading control, the gel was stained with Coomassie R250 (right panel). (C) Calculations of the relative quantities of mitochondrial translation products shown in (B). The frame marks the partial restoration of the ATP6 amount in mutant cells as a result of the MTIF3 synthesis from the plasmid. The error bars were built based on three independent experiments.

Figure 4

The proposed model explaining the effect of MTIF3 deletion in human cells. (A) The situation in the wild type cells when MTIF3 is expressed. (B) The situation in MTIF3-deficient cells. See Discussion for the details.