Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate

Abstract

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

Fig. 1. Tpc1p catalyzes the transport of ThPP and ThMP. (A) Expression of yeast Tpc1p in E.coli. Proteins were separated by SDS–PAGE and stained with Coomassie Blue. The positions of the markers (bovine serum albumin, carbonic anhydrase and cytochrome c) are shown on the left. Lanes 1–4: E.coli CO214(DE3) containing the expression vector with (lanes 2 and 4) and without (lanes 1 and 3) the coding sequence of Tpc1p. Samples were taken at the time of induction (lanes 1 and 2) and 5 h later (lanes 3 and 4). The same number of bacteria was analyzed in each sample. Lane 5: purified Tpc1p (2 µg) originating from bacteria shown in lane 4. (B) Substrate specificity of Tpc1p. Proteoliposomes were pre-loaded internally with various substrates (concentration, 10 mM). Transport was started by external addition of 0.2 mM [α-³⁵S]dATP and stopped after 30 min. (C) Efflux of [α-³⁵S]dATP from proteoliposomes reconstituted with Tpc1p. Proteoliposomes were reconstituted in the presence of 1 mM dATP, 30 mM MES/30 mM HEPES (buffer A) at pH 6.0. The internal substrate pool was labeled by carrier-mediated exchange equilibration. Then the proteoliposomes were passed through Sephadex G-75 columns pre-equilibrated with 50 mM NaCl and 0.1 mM MES/0.1 mM HEPES pH 6.0. The efflux of [α-³⁵S]dATP was started by adding buffer A at pH 6 (filled squares), buffer A at pH 8.0 (filled circles), buffer A at pH 8.0 with 20 mM bathophenanthroline and 60 µM p-CMBS (open circles) or buffer A at pH 8.0 with 10 mM ThPP (filled triangles). (D and E) Dependence on trans-membrane pH gradient of the dATP uniport by Tpc1p. The reconstitution mixture contained 10 mM dATP (dATP/dATP exchange) or 10 mM NaCl (dATP uniport), and buffer A at pH 8.0 (D) or at various pH values from 6.0 to 8.0 (E). After reconstitution of Tpc1p into liposomes, a mixture of 50 mM NaCl and 0.1 mM MES/0.1 mM HEPES at pH 8.0 (D) or various pH values from 6.0 to 8.0 (E) was used to equilibrate and to elute the Sephadex G-75 columns. Transport was started by adding 1 mM [α-³⁵S]dATP together with buffer A at pH values from 6.0 to 8.0 (D) or at pH 6.0 (E). The reaction was terminated after 3 min: dATP uniport (filled squares), dATP/dATP exchange (filled circles). Similar results were obtained in three independent experiments. (F) Effect of externally added substrates on the uptake of [α-³⁵S]dATP into proteoliposomes reconstituted with recombinant Tpc1p. Transport was started by adding 0.2 mM [α-³⁵S]dATP to proteoliposomes containing 10 mM NaCl and no substrate, and stopped after 3 min. All substrates were added together with [α-³⁵S]dATP at a final concentration of 0.8 mM. The extents of inhibition (%) from a representative experiment are reported.

Fig. 2. tpc1Δ mutant is auxotrophic for thiamine. (A) Growth behavior of tpc1Δ in various conditions. Four-fold serial dilutions of wild-type and tpc1Δ mutant strains were plated on solid SM supplemented with 2% glucose. Where indicated, 0.4 mg/l thiamine, 4 mg/ml HET, 4 mg/ml thiazole, 340 µg/ml valine (VAL) or 430 µg/ml isoleucine (ILE) was also present. (B) The expression level of Tpc1p is independent of the presence of thiamine in the SM medium. Equal amounts of mitochondrial lysates from wild-type cells grown on galactose-supplemented SM with or without thiamine (Th) were separated by SDS–PAGE, transferred to nitrocellulose and immunodecorated with the antiserum directed against Tpc1p.

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.

Fig. 4. Comparison of the expression of Tpc1p on various carbon sources. Cells were harvested from exponentially growing cells on YP medium supplemented with the indicated carbon sources. Glucose* indicates cells harvested after diauxic shift. The amount of Tpc1p protein was estimated by densitometry upon immunodecoration of mitochondrial protein with a specific antiserum. Similar results were obtained in four independent experiments. The amount of Tpc1p present in mitochondria from galactose-fed cells was taken as 100%.

Fig. 5. Tpc1p is an integral protein of the inner mitochondrial membrane. (A) Immunoblot analysis of Tpc1p in yeast mitochondria. Equal amounts of mitochondrial lysates from wild-type (lane 1) and tpc1Δ mutant (lane 2) were separated by SDS–PAGE, transferred to nitrocellulose and immunodecorated with antibodies directed against Tpc1p and the ADP/ATP carrier (Aac2p). (B) Tpc1p is not extracted from mitochondrial membranes by carbonate treatment. Proteins of pellets were analyzed by SDS–PAGE and western blotting using antisera directed against Tpc1p, Aac2p (inner membrane component), Tom40p (outer membrane component), cytochrome b₂ (Cyt b₂; intermembrane space marker) and mitochondrial hsp70 (mt-hsp70; matrix protein). The total amount of protein in the pellet and supernatant was set to 100%. (C) Digitonin fractionation of mitochondria. Tpc1p, Aac2p and Tom40p were estimated by immunoblotting. Their content in the unextracted membrane fraction was taken as 100%.