Targeting and insertion of the cholesterol-binding translocator protein into the outer mitochondrial membrane

Abstract

Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP.

Figure 1. Identification of TSPO import complexes in the OMM

A) Isolated mitochondria from HeLa cells were incubated with in vitro-transcribed/translated [³⁵S]-TSPO. The BN-PAGE gel was transferred to PVDF and exposed to a multipurpose phosphor screen (left panel) or blotted with antisera against TSPO, Tom 40, and Tom22 (right panel). B) In vitro-transcribed/translated TSPO import into mitochondria examined at either 4 °C or 33 °C; at the stated time points, one-half of the import reaction was treated with proteinase K and both samples were analyzed by BN-PAGE. C) In vitro-transcribed/translated [³⁵S]-TSPO and [³⁵S]-Tom70 were incubated with HeLa cell mitochondria and treated with sodium carbonate for 30 minutes.

Figure 2. TSPO import is dependent upon heat shock proteins and ATP for import

A) Effects of C90 incubation with TSPO. TSPO was imported as described above (control). B) Effect of ATP depletion, 18μM geldanamycin, and 1mM novobiocin on TSPO import. C) Quantification of imported TSPO. Results shown are means±sem from three independent experiments. D) Nickel-sepharose pull-down assay of ³⁵S-TSPO and ³⁵S-PIC. When indicated geldanamycin or novobiocin were added to the reaction mixture.

Figure 3. Identification of the TSPO amino acid sequence(s) responsible for targeting the protein to the OMM

MA-10 cells were transfected with either wild-type GFP-TSPO or various constructs of GFP-TSPO, stained with Mitotracker CMX and visualized by confocal microscopy. Each panel shows the construct used, GFP fluorescence, mitochondrial staining, and the merged image. A) Wild-type TSPO with GFP linked to the N-terminus. Cells were also transfected with GFP-Δ1−7-TSPO (B), GFP- Δ1−28-TSPO (C), GFP-Δ1−48-TSPO (D), GFP- Δ1−68-TSPO (E), GFP- Δ1−85-TSPO (F), GFP- Δ1−110-TSPO (G), GFP-Δ151−169-TSPO (J), GFP-Δ157−169-TSPO (K). The cholesterol binding domain, GFP-Δ148−157-TSPO (L), or the Schellman motif, GFP-Δ103−108-TSPO (H) were removed and transfected. Construct GFP-G106A-TSPO (I) point mutation altering the Schellman motif and constructs GFP-Leucine^112−114-TSPO (M) and GFP-Leucine^137,138,141-TSPO (N) were also transfected as described under experimental methods.

Figure 4. TSPO protein regions and amino acids required for insertion into the OMM

A) Model of the 18-kDa TSPO protein present in the OMM. Blue amino acids represent the C-terminus, red amino acids represent the mutated leucine residues, and green amino acids show the Schellman motif with the yellow amino acid in the middle representing the mutated glycine. B) Schellman motif mutants of the TSPO protein were compared with wild-type TSPO for import into HeLa cells mitochondria, followed by analysis by BN-PAGE. C) Effect of removal of the C-terminus with constructs Δ157−169 and Δ151−169 on inhibition of TSPO import into isolated mitochondria. D) Effect of leucine mutations on in vitro-transcribed/translated radiolabeled TSPO import into HeLa cell mitochondria. E) SDS-PAGE of radiolabeled constructs from import studies for confirmation of size due to mutations. F) Measurement of progesterone production produced by MA-10 was performed as stated in the methods. Results shown are means ± SEM from three independent experiments (n = 9).

Figure 5. TSPO import is dependent on Metaxin 1

A) MA-10 mitochondria incubated with in vitro-transcribed/translated TSPO analyzed by BN-PAGE, and blotted with anti-metaxin 1 antibody. B) In vitro-transcribed/translated TSPO incubated with mitochondria isolated from MA-10 cells control, Metaxin 1-depleted, and MA-10 cells treated with scrambled siRNAs. C) Immunoblot analysis for Metaxin1, COX IV, TSPO and VDAC in mitochondria isolated form MA-10 cells treated with the various siRNAs. D) Quantification of imported TSPO. Results shown are means±sem from three independent experiments.

Figure 6. TSPO import is increased with cAMP stimulated mitochondria

A) Mitochondria isolated from MA-10 cells incubated with +/− 1mM cAMP were incubated with radiolabeled TSPO, analyzed via BN-PAGE. B) Quantification of imported TSPO, results shown are means±sem from three independent experiments. C) RT-qPCR analysis of MA-10 RNA +/− 1mM cAMP. D) Immunoblot analysis of MA-10 cells incubated with and without 1mM 8-bromo-AMP.

Figure 7. TSPO import into the OMM

We have outlined the steps necessary for TSPO targeting and insertion into the OMM. (1) TSPO is targeted to the OMM through its interactions with cytosolic chaperones, Hsc70 and Hsp90. (2) At the OMM, TSPO interacts with Tom70 and is released from the chaperones in an ATP-dependent manner. (3) TSPO insertion into the OMM is dependent upon Metaxin 1 (Mtx1) and TSPO structure, where both the C-terminus and the Schellman motif (amino acids 103−108) are necessary to form a 66-kDa complex. Once import is complete, TSPO associates with other protein complexes found in the OMM identified previously as VDAC and ANT. Yellow arrow indicates possible step accelerated by cAMP treatment.