Fig. 1
Import of MPC precursors into the mitochondria. a Membrane topology of substrates of the carrier translocase TIM22 in the inner mitochondrial membrane (IM). Left, all TIM22 substrates known so far possess a uniform topology with an even number of transmembrane segments and both termini facing the intermembrane space (IMS): canonical mitochondrial carriers (black) and translocase components (green). Right, the mitochondrial pyruvate carrier subunits Mpc2 and Mpc3 possess an odd number of transmembrane segments and expose the N-terminus to the matrix. The N-terminus of Mpc1 is also located in the matrix, Mpc1 likely possesses three transmembrane segments like Mpc2/3. b Overview of the presequence (TIM23) pathway and the carrier (TIM22) pathway to the IM. Precursors with N-terminal presequence are recognized by the receptor Tom20, translocated through the TOM complex, and transferred to TIM23 for sorting to the IM or matrix. Precursors of the mitochondrial carrier family are recognized by the receptor Tom70, translocated through TOM, and handed over to small TIM chaperones in the IMS (TIM9·10, TIM8·13); the precursors are inserted into the IM by the TIM22 complex. Δψ, membrane potential across the IM; PAM, presequence translocase-associated motor. c Mpc2 and Mpc3 precursors radiolabeled with [35S] methionine were imported at 25 °C into isolated yeast wild-type mitochondria for the indicated periods. Non-imported precursors were degraded with proteinase K (PK) where indicated; the mitochondria were analyzed by SDS-PAGE and autoradiography. d Mpc2 and Mpc3 import and assembly into a native complex is Δψ-dependent. Radiolabeled Mpc2 and Mpc3 precursors were imported as in c; mitochondria were PK-treated and analyzed by BN-PAGE and autoradiography. e Mpc2 and Mpc3 are not proteolytically processed upon import into mitochondria. Mpc2 and Mpc3 were imported into mitochondria as in c. The reactions were analyzed by SDS-PAGE and autoradiography. For comparison, 20% of reticulocyte lysate (precursor) used in the import reactions were included