The human mitochondrial translation factor TACO1 alleviates mitoribosome stalling at polyproline stretches

Abstract

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Graphical Abstract

Figure 1.

Loss of TACO1 causes cytochrome c oxidase deficiency due to aberrant translation of COX1 mRNA. (A) Steady-state levels of TACO1, cytochrome c oxidase subunits (COX1, COX2), F₁F_o-ATP synthase subunit ATP5A, and β-ACTIN in WT, TACO1-KO and TACO1-KO rescued HEK293T cells. (B) BN-PAGE analysis of MRC complexes IV and II in WT, TACO1-KO and TACO1-KO rescued HEK293T cells. (C) Metabolic labeling of newly synthesized mitochondrial translation products with ³⁵S-methionine in the presence of emetine to inhibit cytoplasmic protein synthesis in WT, TACO1-KO and TACO1-KO rescued HEK293T cells. Aberrant products of mitochondrial translation observed in TACO1-KO cells are highlighted with asterisks. Immunoblot analysis against β-ACTIN is provided as a loading control. (D) Densitometric quantification of the newly synthesized mtDNA-encoded proteins. Data are plotted as mean ± SD (n = 3 biological replicates, two-way ANOVA with Dunnet's multiple comparisons, ****P ≤ 0.0001, **P ≤ 0.01). (E) Distribution profiles of ³⁵S-labeled products of mitochondrial protein synthesis are shown in (C). Profiles show the average distribution of the triplicate per each genotype. (F) Transcript levels of mtDNA-encoded genes, normalized by ACTIN mRNA (ACT) levels. Data are plotted as mean ± SD (n = 3 biological replicates, two-way ANOVA with Sidak's multiple comparisons, **P ≤ 0.01). (G) Schematic representation of the puromycin mechanism of action. The top panels show the chemical structures of the tyrosyl-tRNA end and puromycin. The bottom panels illustrate that during protein synthesis, puromycin gets incorporated in an unspecific amino-acid manner and causes translation termination with the release of the nascent polypeptide chain. (H) Puromycin release experiment. Labeling of mitochondrial translation polypeptides with ³⁵S-methionine in WT and TACO1-KO cells as in Figure 1C but for each cell line, in the absence (–) or presence of puromycin during the translation assay. Puromycin was added at the beginning of the ³⁵S-methionine labeling (0′) or after increasing times of pulse-labeling (10′, 20′ and 30′). The 35S-methionine pulse-labeling was extended in an additional sample for thirty additional minutes (+30′). The main puromycin-released translation products are highlighted with an asterisk. Immunoblot analysis against β-ACTIN is provided as a loading control.

Figure 2.

TACO1 binds to the mitoribosome and positively regulates mitochondrial protein synthesis by alleviating polyproline-induced mitoribosome stalling. (A) SILAC-based quantitative proteomic analysis of TACO1 interactors in native conditions. Enriched proteins were determined by applying a t-test, further calculating FDR < 0.05 using Benjamini-Hochberg correction. Red = TACO1-FLAG (bait) is marked in red. Enriched mtLSU ribosome proteins and the OXA1L insertase are highlighted in blue and green colors, respectively. (B) Prey specificity graph for BioID proximity interactome of TACO1 protein, where prey specificity was determined as the relative enrichment of interaction of individual preys and TACO1, compared to their interaction with 100 other mitochondrial baits. Enriched mtLSU ribosome proteins and the OXA1L insertase are highlighted in the same colors as in panel (A). mtSSU proteins and mitoribosome-associated proteins are color-coded in purple and orange, respectively. (C) Relative occupancy of mitoribosomes on COX1 transcript in WT and TACO1-KO cells analyzed by ribosome profiling experiments. Read counts at each codon position were normalized to the total number of read counts for COX1. Proline-rich regions of more than two prolines per 5 amino acid sequence length are highlighted. (D) Relative ribosome occupancies in regions having 0–3 prolines per 5 amino acid sequence length. Ribosome occupancies in TACO1-KO and TACO1 reconstituted cells were normalized to WT cells. (E) Relative distribution of polyproline motifs in human and yeast mtDNA-encoded proteins.

Figure 3.

TACO1 and bL27m cooperate to stabilize the PTC during mitochondrial translation. (A) Cryo-EM structures of the translating mitochondrial ribosome (6ZM5) (114). P-site tRNA is depicted in black; bL27m is depicted in green; mRNA in purple; nascent chain (NC) in orange; 16S rRNA in light blue. Images were prepared in UCSF ChimeraX (115). (B) Metabolic labeling of newly synthesized mitochondrial translation products with ³⁵S-methionine in WT and TACO1-KO cells carrying the empty vector (EV) or overexpressing either WT or bL27m variants carrying mutations (K34A, S35A, S35F or S46F) in its N-terminus. Immunoblot analysis against β-ACTIN is provided as a loading control. (C) Densitometric quantification of the newly synthesized mtDNA-encoded proteins in the genotypes reported in panel (B). Data are plotted as mean ± SD (n = 3 biological replicates, one-way ANOVA with Dunnet's multiple comparisons, *P ≤ 0.05). (D) Low-resolution model of the TACO1 function.