Identification of new channels by systematic analysis of the mitochondrial outer membrane

Abstract

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

Figure 1.

Screen for channels linked to the yeast mitochondrial outer membrane. (Lanes 1–3) Mitochondrial outer membrane (OM) vesicles (OMVs) from por1Δ and WT yeast and mitochondria (Mito.) were analyzed by SDS-PAGE and Coomassie staining. (Lanes 4–6) Crude and purified mitochondria and outer membrane vesicles from por1Δ were analyzed by SDS-PAGE and immunodetection. (Lanes 7–11) Proteins were recombinantly expressed, purified, and analyzed by SDS-PAGE and Coomassie staining. IM, inner membrane.

Figure 2.

Characterization of the channel activities OMC7 and OMC8. (A) Current recordings of the OMC7 channel reconstituted into liposomes in symmetrical (250 mM KCl cis/trans) buffer conditions at the indicated voltage amplitude (V_m). (B) Current-voltage recordings of OMC7 in symmetrical buffer conditions. Mean values of three independent experiments with SD. (C) Current-voltage recordings of OMC7 in asymmetric (250 mM/20 mM KCl cis/trans) buffer conditions. (D) Current recordings of the OMC8 channel reconstituted into liposomes in symmetrical buffer conditions. (E) Expanded current recording from the top left panel in D. (F) Current-voltage recordings of OMC8 in symmetrical buffer conditions. (G) Current-voltage recordings of OMC8 in asymmetric buffer conditions.

Figure 3.

Mim1 forms a channel. (A) Current recordings in symmetrical buffer conditions from a bilayer containing active Mim1 channels at the indicated voltage (V_m). (B) Current-voltage recordings of the Mim1 channel in symmetrical buffer conditions. (C) Mean variance plot of a single Mim1 channel unit calculated from the symmetrical current recording (A) at V_m = 120 mV. (D) Current-voltage recordings of the Mim1 channel without (black) or after coreconstitution with Mim2 (red). Mean values of V_rev of five (Mim1) and four (Mim1 + Mim2) independent experiments with SD (unpaired t test). (E) Mean of the maximal current of the Mim1 channel in the absence (n = 15) and presence (n = 11) of Mim2.

Figure 4.

Ayr1 forms an NADPH-regulated channel. (A) Purified outer membrane vesicles (OMVs) and mitochondria (Mito.) from WT and ayr1Δ yeast were analyzed by SDS-PAGE and Coomassie staining. (B) Fractions enriched for mitochondria (P13) or microsomes (P100) and outer membrane vesicles were analyzed by SDS-PAGE and immunodetection. (C and E) Current recordings of the Ayr1 (404) channel in symmetrical (250 mM KCl cis/trans) buffer conditions at V_m = 140 mV in the absence (C) or presence (E) of 1 mM NADPH. (D and F) Mean variance plot of the Ayr1 channel in the absence (D) or presence of NADPH (F) at V_m = 140 mV. (G) Current-voltage recordings of the Ayr1 channel in symmetrical buffer conditions in the absence (black) or presence of 1 mM NADH (blue) or 1 mM NADPH (red). (H) Gating frequency of the Ayr1 channel in symmetrical buffer conditions in the absence (black) or presence of 1 mM NADPH (red). Mean values of three independent experiments with SD. (I) Open probability of the Ayr1 channel in symmetrical buffer conditions in the absence (black) or presence of 1 mM NADPH (red; n = 3). (J) Current-voltage recordings of the Ayr1 channel in asymmetric (250 mM/20 mM KCl cis/trans) buffer conditions in the absence (black) or presence of 1 mM NADPH (red). (K) Current-voltage recordings of the Ayr1 channel in symmetrical buffer conditions in the absence (black) or presence of 1 mM acyl-dihydroxyacetone-phosphate (acylDHAP; red). (L) Current-voltage recordings of the Ayr1_G20,22A channel in symmetrical buffer conditions in the absence (black) or presence of 1 mM NADH (blue) or 1 mM NADPH (red).