Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

Abstract

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.

FIGURE 1:

MINOS interacts with outer membrane protein complexes. Whole-cell digitonin extracts from wild-type (WT) cells and cells expressing protein A fusion constructs (Fcj1_ProtA and Mio27_ProtA) were subjected to IgG affinity chromatography, elution with TEV protease, and analysis by SDS–PAGE and immunoblotting. Load, 1.5%; elution, 100%. Mio27′, TEV-cleaved form of Mio27_ProtA; OM, outer mitochondrial membrane.

FIGURE 2:

Interaction between MINOS and SAM depends on the POTRA domain of Sam50. Whole-cell powder from wild-type (WT), Sam37_ProtA, and Sam37_ProtA Sam50_Δ120 cells was solubilized in digitonin-containing buffer, subjected to IgG affinity chromatography, and analyzed by SDS–PAGE and immunoblotting. Load, 1%; elution, 100%. OM, outer mitochondrial membrane.

FIGURE 3:

MINOS independently interacts with outer membrane TOM and SAM complexes. Whole-cell digitonin extracts from wild-type (WT), Fcj1_ProtA, and Fcj1_ProtA Sam50_Δ120 cells were subjected to IgG affinity chromatography and analyzed by SDS–PAGE and immunoblotting. Load, 1.5%; elution, 100%. OM, outer mitochondrial membrane.

FIGURE 4:

Deletion of FCJ1, but not of other MINOS components, leads to impaired Tom40 biogenesis. [³⁵S]-labeled Tom40 was imported into mitochondria isolated from wild-type (WT), fcj1Δ, mio10Δ, mio27Δ, aim37Δ, aim5Δ, and aim13Δ cells. Mitochondria were solubilized with digitonin and subjected to blue native electrophoresis and digital autoradiography. Int-I, precursor-SAM assembly intermediate I; Int-II, assembly intermediate II.

FIGURE 5:

Biogenesis of Tom40 is impaired upon depletion of Fcj1. (A) Fcj1↓ (YPH499 fcj1::kanMX6, PGAL1-FCJ1) and wild-type control cells were precultured in the presence of 2% galactose. Subsequently, expression of PGAL1-FCJ1 was inhibited with 1% glucose, mitochondria were isolated, and the mitochondrial membrane potential was assessed using the potential-sensitive dye DiSC₃(5). (B) The [³⁵S]-labeled precursors of F₁-ATPase subunit β (F₁β) and cytochrome c₁ (Cyt. c₁) were imported into isolated mitochondria for the indicated time periods. After proteinase K treatment to remove nonimported precursors, samples were analyzed by SDS–PAGE and digital autoradiography. p, precursor; i, intermediate; m, mature. (C) [³⁵S]ADP/ATP carrier (AAC) or (D) [³⁵S]Tom40 was imported into isolated mitochondria as indicated and analyzed by blue native electrophoresis and digital autoradiography. Int-I, precursor-SAM assembly intermediate I; Int-II, assembly intermediate II.

FIGURE 6:

Mitofilin/Fcj1 is required at an early stage of Tom40 biogenesis. (A) [³⁵S]-labeled Tom40 was imported into Sam37_ProtA and Sam37_ProtA Sam50_Δ120 mitochondria; this was followed by solubilization in digitonin buffer, blue native electrophoresis, and autoradiography. Int-I, precursor-SAM assembly intermediate I; Int-II, assembly intermediate II. (B) Radiolabeled Tom40 was incubated with wild-type (WT) and fcj1Δ mitochondria. Nonimported precursor was removed by proteinase K treatment, and mitochondria were subjected to SDS–PAGE and digital autoradiography. (C) Tom40 import experiments were performed as described in (B) and quantified. Data are represented as mean ± SE of the mean (n = 3), with the exception of the 10-min time point (n = 2; error bar represents range). The amount of protease-protected [³⁵S]Tom40 after 15-min import into wild-type mitochondria was set to 100% (control).