Figure 4.

Occlusion of the poly(A) tail inhibits mitochondrial translation. (A) In vivo mitochondrial translation assays were performed for 15 min (lanes 1 and 2) and 45 min (lanes 3 and 4) exposure to ³⁵S methionine/cysteine after expression of mtPABP1 (lanes 2 and 4) or mtLUC (lanes 1 and 3) for 5 days as described in ‘Materials and Methods’ section. Cell lysates (50 µg) were separated through a 15% denaturing PAG, visualized and quantified using ImageQuant software following exposure to a PhosphorImager cassette. Individual polypeptides were designated on the basis of their mobility (20). To confirm equal loading, a small section of the gel is shown after exposure following Coomassie staining. (B) mtPABP1 and variants are expressed and imported into mitochondria with equal efficiency. Transfectants were induced for 3 days, mitochondria isolated and proteinase K treated as described in ‘Materials and Methods’ section. Resulting mitochondrial lysates (10 µg) were separated by 12% denaturing SDS-PAGE. Western blots were performed with anti-FLAG antibody to detect mtPABP1 and variants. Equal loading was confirmed using two antibodies specific to mitochondrial proteins, the outer membrane protein VDAC (porin) and the soluble matrix marker translation initiation factor 3 (IF3mt). Molecular weight size markers are indicated. (C) Similar in vivo labelling experiments were performed (10-min pulse) on the indicated transfectants after 3 days induction. Lysate (25 µg) was separated and visualized as described in (A). Lane 3, variant mtPAPB1(Y56V/F142V); lane 4, variant mtPABP1(F337V). (D) Induction of mtPAPB1 and variant mtPABP1(F337V) do not increase the turnover of mt-mRNA. RNA was isolated from the indicated transfectants after 0-, 3-, 5- or 8-day induction and aliquots (10 µg) subjected to northern blot analysis as detailed in ‘Materials and Methods’ section. The indicated mt-mRNA probes were used. A probe to the 12 S mt-rRNA (MTRNR1) was used as loading control.