Fig 1: Effect of FASTK family protein depletion on mitochondrial translation, respiratory chain assembly and oxygen consumption.(A) Levels of de novo mitochondrial protein synthesis were measured in WT (control) and FASTKDs-KO HAP1 cells by pulse incorporation of 35S-labeled methionine and cysteine. Equal amounts of whole cell lysates were separated by SDS-PAGE and visualized by autoradiography. (B) Protein expression levels of specific subunits of each of the MRC complexes in WT (control) and FASTKDs-KO cells were evaluated by immunoblotting. Total OXPHOS Rodent WB Antibody Cocktail antibody was used to assess the efficiency of the MRC complex assembly. VDAC1 was included as a mitochondrial loading control. (C) Relative abundance of each assembled MRC complex was determined by quantification of the results of immunoblotting (n = 3). Loading amounts were normalized relative to VDAC1. The dotted line shows the relative abundance of control (= 1). Results are mean±SEM, *P<0.05, **P 0.001 to 0.01, ***P <0.001 (two-tailed unpaired t-test). (D) Oxygen consumption rate (OCR) in WT (control) and FASTKDs-KO cells was measured using the Seahorse analyzer. OCR was normalized to the number of cells in each well. A representative result from three independent biological experiments is shown.
Fig 2: Generation of HAP1 cell lines carrying single or combined knockouts of FASTK family members.(A) Overview of the FASTK protein family and their proposed roles in mitochondrial post-transcriptional regulation. All proteins share a conserved C-terminal domain architecture of FAST_1, FAST_2, and the RAP domains. An internal translation start site exposes a mitochondrial targeting sequence (MTS) leading to expression of the mitochondrial FASTK (mtFASTK). (B) Expression levels of FASTK family proteins and mtRBPs involved in the post-transcriptional regulation in WT (control) and FASTKDs-KO HAP1 cells were measured by immunoblotting. γ-tubulin was used as a loading control for whole cell lysates. mtHSP70 and VDAC1 were used as mitochondrial loading controls. A representative result from two independent experiments is shown.
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