The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

Abstract

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.

Figure 1.

Import of Ugo1 into mitochondria involves Tom70. (A, top) A schematic representation of Ugo1. Transmembrane α helices are marked in gray. (middle) ³⁵S-Ugo1 was imported into wild-type (WT) and Tom40_HA mitochondria (Mito.) for 5 min at 25°C. Mitochondria were lysed with digitonin and subjected to coprecipitation with HA-specific antibodies followed by SDS-PAGE and autoradiography. Load, 2%; elution, 100%. (bottom) Wild-type and Tom40_HA mitochondria were treated as described above and analyzed by SDS-PAGE and Western blotting. Load, 5%; elution, 100%. (B) Chemical amounts of Ugo1_His were imported into wild-type mitochondria for 10 min at 25°C. Mitochondria were lysed with digitonin and subjected to Ni²⁺-NTA agarose purification, blue native electrophoresis, and Western blotting. Load, 5%; elution, 100%. (C–F) ³⁵S-Ugo1 or porin was imported into isolated mitochondria and analyzed by blue native electrophoresis and autoradiography.

Figure 2.

Assembly of multispanning outer membrane proteins is inhibited in the absence of Mim1. (A) Mitochondria (Mito.) were analyzed by SDS-PAGE and Western blotting. WT, wild type. (B and C) ³⁵S-Ugo1 or Scm4 was imported into wild-type and mim1Δ mitochondria. Mitochondria were lysed with digitonin and analyzed by blue native electrophoresis.

Figure 3.

Mim1 promotes the import of Ugo1. ³⁵S-Ugo1 was imported into mitochondria (Mito.) isolated from wild-type (WT) yeast, mim1Δ yeast, and a mim1Δ strain overexpressing Mim1. Mitochondria were lysed with digitonin and analyzed by blue native electrophoresis.

Figure 4.

Ugo 1 binds to the Mim1 complex. (A) Chemical amounts of Ugo1_His were imported into wild-type (WT) mitochondria (Mito.) for 10 min at 25°C. Mitochondria were lysed with digitonin and subjected to Ni-NTA agarose purification, SDS-PAGE, and immunodecoration. Load, 1%; elution, 100%. (B) Import and purification of Ugo1_His were performed as described in A followed by blue native electrophoresis and Western blotting. Load, 5%; elution, 100%. (C) ³⁵S-Tom20 or Tom40 was imported into wild-type mitochondria and mitochondria from a Mim1-overexpressing strain. Mitochondria were lysed with digitonin and subjected to coprecipitation with Mim1-specific antiserum and SDS-PAGE. Precursor, 5% of reticulocyte lysate.

Figure 5.

Tom70 and Mim1 cooperate in the biogenesis of Ugo1. (A) ³⁵S-Ugo1 was incubated with Tom70_CD and Tom20_CD coupled to Ni-NTA. Bound proteins were eluted and analyzed by SDS-PAGE. Load, 5%; elution, 100%. (B) Chemical amounts of Ugo1_His were imported into mitochondria (Mito.) for 10 min at 25°C. Mitochondria were lysed with digitonin and subjected to Ni-NTA agarose purification, blue native electrophoresis, and Western blotting. Load, 2%; elution, 100%. WT, wild type. (C, top) ³⁵S-Ugo1 was imported into mitochondria for 10 min at 25°C. Mitochondria were lysed with digitonin, and immunoprecipitation with the indicated antisera was performed, followed by SDS-PAGE and autoradiography. Load, 3%; elution, 100%. (bottom) Quantification of three independent experiments with standard error of the means. Coprecipitation of ³⁵S-Ugo1 with anti-Mim1 in wild type was set to 100% (control). PI, preimmune. (D) Mitochondria were solubilized with digitonin and incubated with Ni-NTA agarose. Bound proteins were eluted and subjected to SDS-PAGE and immunodecoration with the indicated antisera. Load, 0.5%; elution, 100%. (E) Mitochondria were subjected to cross-linking with MBS, solubilized with SDS, and incubated with Ni-NTA agarose. Bound proteins were eluted and analyzed by SDS-PAGE and immunodecoration. Arrowhead, unspecific band.