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%.