Role of phosphatidylethanolamine in the biogenesis of mitochondrial outer membrane proteins

Abstract

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.

Keywords: Membrane Biogenesis; Mitochondria; Phospholipid; Protein Targeting; Protein Translocation.

FIGURE 1.

Biogenesis of β-barrel proteins is impaired in PE-depleted mitochondria. A, the phospholipid distribution of isolated WT, psd1Δ, and psd1Δ psd2Δ mitochondria was determined. Means ± S.E. (n = 4) are shown for the mitochondrial preparation. B, the phospholipid distribution of isolated WT, psd1Δ, and psd1Δ psd2Δ outer membrane vesicles was determined. The means with range from two independent experiments are depicted. LP, lysophospholipids; PI, phosphatidylinositol; PS, phosphatidylserine; DMPE, dimethylphosphatidylethanolamine; PA, phosphatidic acid. C, ³⁵S-labeled Tom40 was imported into isolated WT, psd1Δ, or psd1Δ psd2Δ mitochondria at 25 °C for the indicated time periods. The mitochondria were lysed with digitonin and analyzed by blue native electrophoresis and digital autoradiography. SAM, Tom40 precursor bound to the SAM complex; Int-II, second intermediate of the Tom40 assembly pathway. D, quantification of the three assembly steps of Tom40 (import was performed and analyzed as described for C). Means ± S.E. (n = 5) are shown for the formation of the SAM intermediate after 5 min of import, intermediate II after 20 min of import, and the mature TOM complex after 40 min of import. E and F, porin or Mdm10, respectively, was imported into isolated WT, psd1Δ, or psd1Δ psd2Δ mitochondria at 25 °C for the indicated time periods. The mitochondria were lysed with digitonin and analyzed by blue native electrophoresis and digital autoradiography.

FIGURE 2.

Biogenesis of α-helical outer membrane proteins is not inhibited in PE-depleted mitochondria. ³⁵S-Labeled Tom22 (A), Tom20 (B), or Ugo1 (C) was imported into isolated WT, psd1Δ, or psd1Δ psd2Δ mitochondria at 25 °C for the indicated time periods. The mitochondria were lysed with digitonin and analyzed by blue native electrophoresis and digital autoradiography.

FIGURE 3.

Impaired transport of β-barrel precursors across the outer membrane of PE-depleted mitochondria. A, proteins of isolated WT, psd1Δ, and psd1Δ psd2Δ mitochondria were analyzed by SDS-PAGE and detected by immunodecoration with the indicated antisera. B, WT, psd1Δ, and psd1Δ psd2Δ mitochondria were lysed with digitonin and separated by blue native electrophoresis. Protein complexes were detected by immunodecoration with the indicated antisera. C, ³⁵S-labeled Tom40(G354A) was imported into WT, psd1Δ, and psd1Δ psd2Δ mitochondria. The imported proteins were analyzed by blue native electrophoresis and autoradiography. D, ³⁵S-labeled Tom40 was imported into WT, psd1Δ, and psd1Δ psd2Δ mitochondria, followed by proteinase K (Prot. K) treatment as indicated. Proteins were separated by SDS-PAGE.

FIGURE 4.

Depletion of PE does not affect the stability of the TOM complex. A, WT, psd1Δ, psd1Δ psd2Δ, and crd1Δ mitochondria were lysed with digitonin and separated by blue native electrophoresis. Protein complexes were detected by immunodecoration with the indicated antisera. B, WT, psd1Δ, and psd1Δ psd2Δ mitochondria (Mito.) were lysed with digitonin and subjected to co-immunoprecipitation with the indicated antisera. Proteins were eluted, separated by SDS-PAGE, and detected by immunodecoration with the indicated antisera (4% load and 100% elution).

FIGURE 5.

PE is required for precursor binding to the TOM complex. ³⁵S-Labeled Oxa1 was imported into WT, psd1Δ, and psd1Δ psd2Δ mitochondria (Mito.) in the absence of a membrane potential. The samples were analyzed by blue native electrophoresis and autoradiography.