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.