The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis

Abstract

The mitochondrial inner membrane contains different translocator systems for the import of presequence-carrying proteins and carrier proteins. The translocator assembly and maintenance protein 41 (Tam41/mitochondrial matrix protein 37) was identified as a new member of the mitochondrial protein translocator systems by its role in maintaining the integrity and activity of the presequence translocase of the inner membrane (TIM23 complex). Here we demonstrate that the assembly of proteins imported by the carrier translocase, TIM22 complex, is even more strongly affected by the lack of Tam41. Moreover, respiratory chain supercomplexes and the inner membrane potential are impaired by lack of Tam41. The phenotype of Tam41-deficient mitochondria thus resembles that of mitochondria lacking cardiolipin. Indeed, we found that Tam41 is required for the biosynthesis of the dimeric phospholipid cardiolipin. The pleiotropic effects of the translocator maintenance protein on preprotein import and respiratory chain can be attributed to its role in biosynthesis of mitochondrial cardiolipin.

Figure 1.

Protein import and assembly defects in tam41Δ mitochondria. (A) Biogenesis stages I–V of mitochondrial carrier proteins (Ryan et al., 1999). (B) Isolated mitochondria from wild-type (WT) or tam41Δ yeast cells grown at 21°C were heat shocked in vitro and incubated with radiolabeled AAC or DIC precursor proteins at 25°C. The mitochondria were lysed with digitonin and analyzed by blue native electrophoresis and digital autoradiography. *, carrier form in tam41Δ mitochondria. (C) The assembly of AAC was analyzed as for B without in vitro heat shock. (D) After incubation with AAC, mitochondria were treated at pH 11.5. Total (T), pellet (P), and supernatant (S) were analyzed by SDS-PAGE followed by autoradiography and immunodecoration. (E) Isolated mitochondria (in vitro heat shock) were incubated with Fe/S protein. (F) Quantification of Fe/S protein maturation in the absence or presence of an in vitro heat shock in tam41Δ mitochondria. 20-min import into wild-type mitochondria was set to 100%. Error bars represent SEM (n = 3).

Figure 2.

Temperature sensitivity of Δψ generation in tam41Δ mitochondria. (A) Yeast cells were grown in YPLac at low temperature (21°C) and shifted to 30 or 37°C for 8 h in vivo before isolation of mitochondria. Δψ was assessed at 25°C by fluorescence quenching. (B) Yeast cells were grown at 21°C. Mitochondria were isolated and Δψ was assessed.

Figure 3.

Inner membrane complexes are altered in tam41Δ, crd1Δ, and pgs1Δ mitochondria. (A–D and G) Mitochondria isolated from yeast cells grown at low temperature were analyzed by blue native electrophoresis. Whole cell protein extracts (E) and mitochondria (F) were analyzed by SDS-PAGE and immunodecoration.

Figure 4.

tam41Δ mitochondria are deficient in cardiolipin. (A) Lipids were extracted from isolated mitochondria and subjected to LCMS. Averaged normalized spectra from wild-type and tam41Δ mitochondria. Differential lipid profiles (log₁₀ ratio) of tam41Δ/wild-type, crd1Δ/wild-type, and taz1Δ/wild-type mitochondria. IS, internal standard (tetramyristoyl cardiolipin); PE, phosphatidylethanolamine; PI, phosphatidylinositol. (B) Analysis of cardiolipin levels in wild-type and tam41Δ mitochondria. (C) Wild-type and tam41Δ cells harboring the indicated plasmids were subjected to fivefold serial dilutions and grown at 37°C on YPD. (D) Cardiolipin levels of mitochondria from tam41Δ yeast cells harboring the indicated plasmids. Bars represent the range of two MS analyses.

Figure 5.

tam41Δ mitochondria are deficient in PG but accumulate PA. (A) Cellular lipids in wild type and tam41Δ were labeled with [³²PO₄], extracted, subjected to one-dimensional thin layer chromatography, and imaged. CL, cardiolipin; Ori, origin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine. (B) Cellular lipids labeled and extracted as in A were subjected to two-dimensional thin layer chromatography. (C and D) Analysis of PG and PA levels in wild-type, tam41Δ, and crd1Δ mitochondria. (E) PA levels of tam41Δ mitochondria from a yeast strain reexpressing Tam41. Bars represent the range of two MS analyses.