Targeting and insertion of the cholesterol‐binding Translocator Protein into the outer mitochondrial membrane

Translocator Protein (18‐KDa; TSPO) is a high affinity cholesterol‐ and drug‐binding protein, previously known as the peripheral‐type benzodiazepine receptor. TSPO has been shown to assist with the transport of cholesterol from the cytosol to the inner mitochondria membrane; the rate‐limiting step in the production of steroid hormones. To ensure the production of steroids is tightly regulated, the targeting and insertion of TSPO into the outer mitochondrial membrane must be performed correctly. To understand the mechanisms underlying TSPO import, protein targeting through GFP was used; along with BN‐Page to identify protein complexes. The results obtained show that amino acids #103–109, which contain a Schellman motif, are necessary for targeting TSPO to the mitochondria. The results from the BN‐Page analysis showed the 18‐KDaTSPO to be present in 66‐ and 140‐KDa protein complexes. Further analysis of the native gel by mass spectrometry identified voltage‐dependent anion channel (VDAC) and heat shock protein 70 (HSP70) to be present in the 66‐ and 140‐KDa complexes, respectively. Based on these findings we propose that HSP70 targets TSPO to the outer mitochondrial membrane where through proper protein folding, defined by amino acids #103–109, it is inserted into the membrane at sites enriched in VDAC. (Supported by ES07747).