Fig. 3. Tpc1p is required for entry of ThPP into mitochondria. Mitochondria and post-mitochondrial supernatants (PMS) were isolated from wild-type and tpc1Δ cells grown on thiamine-less SM supplemented with galactose (A–D) or ethanol (E–H), or grown on YP (I and J) supplemented with galactose (Gal), ethanol (Et) or lactate (Lac). (A and E) Mitochondria from tpc1Δ cells grown on galactose, but not on ethanol, are defective in ALS activity. Mitochondria from wild-type and tpc1Δ cells were lysed in a mixture containing 0.05% Triton X-114 and pyruvate-less assay buffer for ALS with or without 1 mM ThPP. The extracts were centrifuged at 12 000 g for 10 min at 4°C. The ALS assay at 30°C was started by adding 50 mM pyruvate to the extracts. (B and F) tpc1Δ PMS contain wild-type levels of PDC activity. PMS from wild-type and tpc1Δ cells were pre-incubated with or without 5 mM thiamine at 30°C in a medium containing 75 mM MgCl₂ and 20 mM PIPES pH 7.0. After 3 min, an aliquot of the pre-incubation mixture was added to the assay buffer for PDC. (C, D and G–J) Mitochondria, but not PMS, isolated from tpc1Δ cells grown on galactose (but not on ethanol or lactate), contain decreased levels of ThPP. Mitochondrial extracts (C, G and I) and PMS (D, H and J) from wild-type and tpc1Δ cells were assayed for ThPP content.