9.A.24 The Mitochondrial Cholesterol/Porphyrin/5-aminolevulinic acid Uptake Translocator Protein (TSPO) Family

The central channel Tom40 of the preprotein translocase of the outer mitochondrial membrane (TOM of the OMM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. Otera et al. (2007) analyzed the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic TMSs and functions in cholesterol and porphyrin import into the inner membrane. Analyses of in vitro and in vivo import into TOM component-depleted mitochondria revealed that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 nor Tom22 import receptors, nor the import channel Tom40, (2) shares the post-Tom70 pathway with  C-tail-anchored proteins, and (3) requires factors in the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway (Otera et al., 2007). TSPO, a 5 TMS protein localized to the outer mitochondria membrane, is a high affinity cholesterol-binding protein (Georges et al. 2021). TSPO ligands modulate neuroplasticity and elicit antidepressant and anxiolytic therapeutic effects in animals and humans (Rupprecht et al. 2022). TSPO ligands are promising candidates for post-ischaemic recovery, exerting neuroprotection, in contrast to midazolam, without detrimental effects on synaptic plasticity (Puig-Bosch et al. 2023).

TSPO or PBR is an 18 kDa high affinity cholesterol, porphyrin uptake and drug-binding protein. Although TSPO is found in many tissue types, it is expressed at the highest levels under normal conditions in tissues that synthesize steroids (Batarseh and Papadopoulos, 2010). TSPO, the 3-d structure of which is known (PDB# 2MGY) has been associated with cholesterol import into mitochondria, a key function in steroidogenesis, and directly or indirectly with multiple other cellular functions including apoptosis, cell proliferation, differentiation, anion transport, porphyrin transport, heme synthesis, and regulation of mitochondrial function (Jaremko et al. 2014). Aberrant expression of TSPO has been linked to multiple diseases, including cancer, brain injury, neurodegeneration, Parkinson''s and Alheimer''s diseases, and ischemia-reperfusion injury.  It forms a large complex that includes VDAC-1, TSPO-associated protein-7 (PAP7; ACBD3), a protein kinase regulatory subunit, PKAR1A, and the StAR regluatory protein (Miller 2013).  TSPO is conformationally flexible (Jaremko et al. 2015). More recent studies have led to the conclusion that TSPO2 transports 5-aminolevulinic acid (see 9.A.24.1.17) (Manceau et al. 2020). The interplay of cholesterol and ligand binding in human TSPO (TC# 9.A.24.1.1) has been studied using classical molecular dynamics simulations (Lai et al. 2021).

Neurosteroids are able to rapidly control the excitability of the central nervous system, acting as regulators of type A receptors for GABA. Neurosteroid level alterations occur in psychiatric disorders, including anxiety disorders. Investigators have manipulated neurosteroidogenesis in an effort to correct neuronal excitation and inhibition imbalances, which may lie at the root of neuropsychiatric conditions. A promising target for therapy of anxiety disorders is the Translocator Protein (TSPO). TSPO is expressed predominantly in steroid-synthesizing tissues and is localized to contact sites between the outer and inner mitochondrial membranes.  It may mediate the rate-limiting step of neurosteroidogenesis. Brain concentrations of neurosteroids can be affected by selective TSPO activation. Indeed, TSPO drug ligands are able to stimulate primary neurosteroid formation that enhances GABAA receptor activity, pregnenolone and allopregnenalone, in both in vitro steroidogenic cells and in vivo animal models. A spectrum of TSPO ligands has been shown to exert anxiolytic actions when administered in rodents. The selective TSPO ligand, XBD173 (AC-5216, Emapunil), exerts anxiolytic effects not only in animal models, but also in humans. (Costa et al., 2012) reviewed the literature regarding the central nervous system biology of TSPO.

The TSPO (18 kDa translocator protein) is involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high-affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but they also stimulate other physiological processes including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. TSPO is present in enterocyte mitochondria but not rat intestinal goblet cells (Ostuni et al. 2009). Enterocyte cytoplasm also contains the endogenous TSPO ligand, polypeptide DBI (diazepam-binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from that in the jejunum and ileum. Specifically, benzodiazepine Ro5-4864 and protoporphyrin IX showed 5-13-fold lower affinity for duodenal TSPO. PK 11195 stimulated calcium-dependent chloride secretion in the duodenum and calcium-dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Thus, the functional differences in subpopulations of TSPO in different regions of the intestine could be related to the structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum, respectively.

A 5 TMS bacterial homologue called the tryptophan-rich sensory protein of Rhodobacter spheroides binds retinoic acid, cucumin and an inhibitor of Bcl-2 actioin called gossypol (Li et al. 2013).  It appears to function in porphyrin degredation in a light- and oxygen-dependent process (Ginter et al. 2013). The protein and its function(s) have been reviewed. TSPO is believed to be involved either directly or indirectly in numerous biological functions, including mitochondrial cholesterol transport and steroid hormone biosynthesis, porphyrin transport and heme synthesis, apoptosis, cell proliferation, and anion transport. Localized to the outer mitochondrial membrane of steroidogenic cells, TSPO has been shown to associate with cytosolic and mitochondrial proteins as part of a large multiprotein complex involved in mitochondrial cholesterol transport, the rate-limiting step in steroidogenesis. It has been concluded that TSPO is a unique mitochondrial pharmacological target for diseases that involve increased mitochondrial activity, including steroidogenesis, but the specific function is not clear (Papadopoulos et al. 2017). However, TSPO has been shown to interact with other cellular proteins: VDAC, 30 kDa adenine nucleotide translocase (ANT), cyclophilin D, hexokinase, creatinine kinase, diazepam binding inhibitor (DBI), phosphate carrier and Bcl-2 family proteins (Kołodziejczyk 2015).

TSPO functions in cholesterol import, mitochondrial metabolism, apoptosis, cell proliferation, Ca2+ signaling, oxidative stress, and inflammation. TSPO forms a complex with VDAC, a protein that mediates the flux of ions, including Ca2+, nucleotides, and metabolites across the OMM, and it controls metabolism and apoptosis while interacting with many proteins. Both TSPO and VDAC are over-expressed in brains from Alzheimer's disease patients. TSPO-interacting ligands have been considered as a potential basis for drug development (Shoshan-Barmatz et al. 2019). The Translocator Protein of 18 kDa (TSPO) has an alternative binding site for the benzodiazepine diazepam. It is an evolutionary well-conserved and tryptophan-rich 169-amino acids protein with five alpha helical transmembrane domains stretching the outer mitochondrial membrane, with the carboxyl-terminus in the cytosol and a short amino-terminus in the intermembrane space of mitochondrion. Together with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), it forms the mitochondrial permeability transition pore (MPTP). TSPO expression is ubiquitary, with higher levels in steroid producing tissues; in the central nervous system, it is mainly expressed in glial cells and neurons. TSPO is implicated in a variety of fundamental cellular processes including steroidogenesis, heme biosynthesis, mitochondrial respiration, mitochondrial membrane potential, cell proliferation and differentiation, cell life/death balance, and oxidative stress. Altered TSPO expression has been found in some pathological conditions. In particular, high TSPO expression levels have been documented in cancer, neuroinflammation, and brain injury. Conversely, low TSPO expression levels have been evidenced in anxiety disorders. Therefore, TSPO is not only an interesting drug target for therapeutic purpose (anticonvulsant, anxiolytic, etc.), but also a valid diagnostic marker of related-diseases detectable by fluorescent or radiolabeled ligands. Barresi et al. 2020 have presented an update of previous reviews dealing with the medicinal chemistry of TSPO and highlighted the most outstanding advances in the development of TSPO ligands as potential therapeutic or diagnostic tools. Compounds enhancing GABAergic neurotransmission such as neurosteroids and TSPO ligands, which also may exert anti-inflammatory properties in concert with immunomodulators such as C1q may open new avenues for the treatment of psychiatric disorders (Rupprecht et al. 2021). Together with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocase (ANT), it forms the mitochondrial permeability transition pore (MPTP) (Rupprecht et al. 2021).

TSPO is phylogenetically widespread from archaea and bacteria to insects, vertebrates, plants, and fungi. TSPO's primary amino acid sequence is only modestly conserved between diverse species, although its five transmembrane helical structure appears mainly conserved (Hiser et al. 2021). Its cellular location and orientation in membranes have been reported to vary between species and tissues, with implications for potential diverse binding partners and function. Most TSPO functions relate to stress-induced changes in metabolism; it could be a receptor, a sensor, a transporter, or a translocator or more than one of these. TSPO may act indirectly by association with various protein binding partners or with endogenous or exogenous ligands. Hiser et al. 2021 review proteins that have commonly been invoked as TSPO binding partners. Possibly TSPO was originally a bacterial receptor/stress sensor associated with porphyrin binding as its most ancestral function and that it later developed additional stress-related roles in eukaryotes as its ability to bind new partners evolved.



Asih, P.R., A. Poljak, M. Kassiou, Y.D. Ke, and L.M. Ittner. (2022). Differential mitochondrial protein interaction profile between human translocator protein and its A147T polymorphism variant. PLoS One 17: e0254296.

Austin CJ., Kahlert J., Kassiou M. and Rendina LM. (2013). The translocator protein (TSPO): a novel target for cancer chemotherapy. Int J Biochem Cell Biol. 45(7):1212-6.

Barresi, E., M. Robello, B. Costa, E. Da Pozzo, E. Baglini, S. Salerno, F. Da Settimo, C. Martini, and S. Taliani. (2020). An update into the medicinal chemistry of translocator protein (TSPO) ligands. Eur J Med Chem 112924. [Epub: Ahead of Print]

Batarseh, A. and V. Papadopoulos. (2010). Regulation of translocator protein 18 kDa (TSPO) expression in health and disease states. Mol. Cell Endocrinol 327: 1-12.

Costa, B., E. Da Pozzo, and C. Martini. (2012). Translocator protein as a promising target for novel anxiolytics. Curr Top Med Chem 12: 270-285.

Georges, E., C. Sottas, Y. Li, and V. Papadopoulos. (2021). Direct and specific binding of cholesterol to the mitochondrial translocator protein (TSPO) using PhotoClick cholesterol analogue. J Biochem 170: 239-243.

Ginter, C., I. Kiburu, and O. Boudker. (2013). Chemical catalysis by the translocator protein (18 kDa). Biochemistry 52: 3609-3611.

Guillaumot, D., S. Guillon, T. Déplanque, C. Vanhee, C. Gumy, D. Masquelier, P. Morsomme, and H. Batoko. (2009). The Arabidopsis TSPO-related protein is a stress and abscisic acid-regulated, endoplasmic reticulum-Golgi-localized membrane protein. Plant J. 60: 242-256.

Guo, Y., R.C. Kalathur, Q. Liu, B. Kloss, R. Bruni, C. Ginter, E. Kloppmann, B. Rost, and W.A. Hendrickson. (2015). Protein structure. Structure and activity of tryptophan-rich TSPO proteins. Science 347: 551-555.

Hiser, C., B.L. Montgomery, and S. Ferguson-Miller. (2021). TSPO protein binding partners in bacteria, animals, and plants. J. Bioenerg. Biomembr. 53: 463-487.

Hug, L.A., B.J. Baker, K. Anantharaman, C.T. Brown, A.J. Probst, C.J. Castelle, C.N. Butterfield, A.W. Hernsdorf, Y. Amano, K. Ise, Y. Suzuki, N. Dudek, D.A. Relman, K.M. Finstad, R. Amundson, B.C. Thomas, and J.F. Banfield. (2016). A new view of the tree of life. Nat Microbiol 1: 16048.

Jaremko L., Jaremko M., Giller K., Becker S. and Zweckstetter M. (2015). Conformational Flexibility in the Transmembrane Protein TSPO. Chemistry. 21(46):16555-63.

Jaremko, L., M. Jaremko, K. Giller, S. Becker, and M. Zweckstetter. (2014). Structure of the mitochondrial translocator protein in complex with a diagnostic ligand. Science 343: 1363-1366.

Korkhov, V.M., C. Sachse, J.M. Short, and C.G. Tate. (2010). Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. Structure 18: 677-687.

Kołodziejczyk, A. (2015). [18 kDa translocator protein--implications in cell''s functions]. Postepy Hig Med Dosw (Online) 69: 34-50.

Lai, H.T.T., A. Giorgetti, G. Rossetti, T.T. Nguyen, P. Carloni, and A. Kranjc. (2021). The Interplay of Cholesterol and Ligand Binding in TSPO from Classical Molecular Dynamics Simulations. Molecules 26:.

Li, F., J. Liu, Y. Zheng, R.M. Garavito, and S. Ferguson-Miller. (2015). Protein structure. Crystal structures of translocator protein (TSPO) and mutant mimic of a human polymorphism. Science 347: 555-558.

Li, F., Y. Xia, J. Meiler, and S. Ferguson-Miller. (2013). Characterization and modeling of the oligomeric state and ligand binding behavior of purified translocator protein 18 kDa from Rhodobacter sphaeroides. Biochemistry 52: 5884-5899.

Manceau, H., S.D. Lefevre, A. Mirmiran, C. Hattab, H.R. Sugier, C. Schmitt, K. Peoc'h, H. Puy, M.A. Ostuni, L. Gouya, and J.J. Lacapere. (2020). TSPO2 translocates 5-aminolevulinic acid into human erythroleukemia cells. Biol Cell. [Epub: Ahead of Print]

Miller, W.L. (2013). Steroid hormone synthesis in mitochondria. Mol. Cell Endocrinol 379: 62-73.

Ostuni, M.A., G. Péranzi, R.A. Ducroc, M. Fasseu, B. Vidic, J. Dumont, V. Papadopoulos, and J.J. Lacapere. (2009). Distribution, pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine. Biol Cell 101: 573-586.

Otera, H., Y. Taira, C. Horie, Y. Suzuki, H. Suzuki, K. Setoguchi, H. Kato, T. Oka, and K. Mihara. (2007). A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments. J. Cell Biol. 179: 1355-1363.

Papadopoulos, V., J. Fan, and B. Zirkin. (2017). Translocator protein (18 kDa): an update on its function in steroidogenesis. J Neuroendocrinol. [Epub: Ahead of Print]

Puig-Bosch, X., M. Ballmann, S. Bieletzki, B. Antkowiak, U. Rudolph, H.U. Zeilhofer, and G. Rammes. (2023). Neurosteroids Mediate Neuroprotection in an In Vitro Model of Hypoxic/Hypoglycaemic Excitotoxicity via δ-GABA Receptors without Affecting Synaptic Plasticity. Int J Mol Sci 24:.

Riond, J., M.G. Mattei, M. Kaghad, X. Dumont, J.C. Guillemot, G. Le Fur, D. Caput, and P. Ferrara. (1991). Molecular cloning and chromosomal localization of a human peripheral-type benzodiazepine receptor. Eur J Biochem 195: 305-311.

Rupprecht, R., C. Rupprecht, B. Di Benedetto, and G. Rammes. (2021). Neuroinflammation and psychiatric disorders: relevance of C1q, translocator protein (18 kDa) (TSPO), and neurosteroids. World J Biol Psychiatry 1-20. [Epub: Ahead of Print]

Rupprecht, R., C.H. Wetzel, M. Dorostkar, J. Herms, N.L. Albert, J. Schwarzbach, M. Schumacher, and I.D. Neumann. (2022). Translocator protein (18kDa) TSPO: a new diagnostic or therapeutic target for stress-related disorders? Mol Psychiatry 27: 2918-2926.

Shoshan-Barmatz, V., S. Pittala, and D. Mizrachi. (2019). VDAC1 and the TSPO: Expression, Interactions, and Associated Functions in Health and Disease States. Int J Mol Sci 20:.

Taylor, J.M., A.M. Allen, and A. Graham. (2014). Targeting mitochondrial 18 kDa translocator protein (TSPO) regulates macrophage cholesterol efflux and lipid phenotype. Clin Sci (Lond) 127: 603-613.

Yeliseev, A.A. and S. Kaplan. (1999). A novel mechanism for the regulation of photosynthesis gene expression by the TspO outer membrane protein of Rhodobacter sphaeroides 2.4.1. J. Biol. Chem. 274: 21234-21243.

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TC#NameOrganismal TypeExample

The peripheral benzodiazepine receptor (PBR), which can bind isoquinoline carboxamides (Riond et al. 1991) and integrates into the mitochondrial outer membrane (MOM) through five hydrophobic TMSs. The protein has 7 TMSs in a probable 2 + 1 + 4 TMS arrangement.  It is also called "translocator protein", TSPO. It is a mitochondrial cholesterol and porphyrin uptake transporter (Jaremko et al. 2014; Taylor et al. 2014) but is also part of the mitochondrial permeability transition pore (MPTP) which includes cyclophilin D, VDAC (TC#1.B.8) and the adenine nucleotide translocator (Austin et al. 2013).  The 3-d structure has been determined at 2.4 Å resolution bound to its high affinity ligand, PK11195 which causes the otherwise loose 5 helix bundle to form a tight bundle with a hydrophobic pocket for PK11195 (Jaremko et al. 2014). It is upregulated in glial cells during neuroinflammation in Alzheimer's disease (Asih et al. 2022). The common A147T polymorphism compromises ligand binding and has been linked to increased risk of neuropsychiatric disorders, possibly by reducing TSPO protein stability. WT TSPO binds 30 partners, yet A147T TSPO binds only 23, one of which is 14-3-3 theta (YWHAQ) (TC# 8.A.98.1.9) (Asih et al. 2022).


PBR of Homo sapiens (Q6ICF9)


Uncharacterized protein of 171 aas and 5 TMSs


UP of Loa loa (Eye worm) (Filaria loa)


TspO protein of 141 aas and 4-5 TMSs


TspO of Phaeocystis globosa virus


TspO-like; MBR-like protein of 163 aas and 4 TMSs in a 1 + 3 TMS arrangement.

Red algae

TspO-like protein of Galdieria sulfuraria


TspO/MBR family member of 151 aas and 5 TMSs.  The crystal structure has been determined at 1.7 Å resolution (Guo et al. 2015).  The protein was solved in complex with the benzodiazepine-like inhibitor, PK11195. TspO-mediated protoporphyrin IX (PpIX) reactions were also described, including catalytic degradation to a previously undescribed heme derivative. Structure-inspired mutations allowed the investigation of the reaction mechanisms, showing that TSPOs from Xenopus and man have similar PpIX-directed activities. Although TSPOs have been regarded as transporters, the catalytic activity in PpIX degradation suggests physiological importance for TSPOs in protection against oxidative stress (Guo et al. 2015).


TspO of Bacillus cereus


Uncharacterized protein of 134 aas and 4 TMSs (Hug et al. 2016).

UP of Candidatus Peribacter riflensis


Uncharacterized protein of 161 aas and 4 TMSs.

UP of Luteimonas mephitis


Tryptophan-rich sensory protein. TSPO, of 167 aas and 5 TMSs

TSPO of Halococcus sediminicola


TSPO2 of 170 aas and 5 TMSs in a 1 + 4 TMS arrangement.  5-Aminolevulinic acid (ALA) is the first precursor of heme biosynthesis pathway. The exogenous addition of ALA to cells leads to protoporphyrin IX (PPIX) accumulation. Several types of ALA transporters have been described depending on the cell type, but there was no clear entry pathway for erythroid cells. The 18 kDa translocator protein (TSPO) has been proposed to be involved in the transport of porphyrins and heme analogs, but ALA-induced PPIX accumulation in erythroleukemia cells (UT-7 and K562) was impaired by PK 11195, a competitive inhibitor of both transmembrane proteins TSPO (1 and 2). PK 11195 did not modify the activity of the enzymes of heme biosynthesis, suggesting that ALA entry at the plasma membrane is the limiting factor. In contrast, porphobilinogen (PBG)-induced PPIX accumulation was not affected by PK 11195, suggesting that plasma membrane TSPO2 is a selective transporter of ALA. Overexpression of TSPO2 at the plasma membrane of erythroleukemia cells increased ALA-induced PPIX accumulation, confirming the role of TSPO2 in the import of ALA into the cells. Thus, ALA-induced PPIX accumulation in erythroid cells involves TSPO2 as a selective translocator through the plasma membrane (Manceau et al. 2020).

TSPO2 of Homo sapiens


The outer membrane tryptophan-rich sensory protein (TspO) of the TSPO/MBR family of 159 aas and 5 TMSs (Yeliseev et al. 1997; Yeliseev and Kaplan 1999).  The 10 Å cryo electron microscopy structure is known (Korkhov et al. 2010) as are 1.8, 2.4 and 2.5 Å structures solved by x-ray crystallography (Li et al. 2015).  Crystals obtained in the lipidic cubic phase reveal the binding site of an endogenous porphyrin ligand. The three crystal structures reveal a dimer, providing insights into the controversial physiological role of TSPO and how a mutation in the human homologue causes psychiatric disorders and reduced pregnenolone production (Li et al. 2015).


TspO of Rhodobacter spheroides


The Endoplasmic reticulum/Golgi TSPO protein is mainly detected in dry seeds, but can be induced in vegetative tissues by osmotic or salt stress or abscisic acid (ABA) treatment (Guillaumot et al. 2009).


TSPO of Arabidopsis thaliana



TspO of Nostoc sp.

 TspO/MBR family protein of 186 aas and 5 TMSs


 TspO/MBR family protein of Acanthamoeba castellanii


TspO of Niastella koreensis


TspO of 159 aas and 5 TM


TspO of Lactococcus lactis


Peripheral-type benzodiazepine receptor of 188 aas and 4 or 5 TMSs.


Peripheral-type benzodiazepine receptor of Zea mays


TspO homologue of 193 aas and 4 or 5 TMSs.


TspO of Oryza sativa


TC#NameOrganismal TypeExample

TspO homologue of 171 aas and 5 TMSs in a 1 + 4 TMS arrangement.


TspO homologue of Maricaulis maris


CrtK protein of 166 aas and 5 TMSs.


CrtK of Oceanicaulis sp.


TC#NameOrganismal TypeExample

Uncharacterized protein of 177 aas and 5 probable TMSs


UP of Mycobacterium vanbaalenii


Tryptophan-rich sensory proteinof 160 aas and 5 TMSs

TpsO of Nocardia soli


TC#NameOrganismal TypeExample

Uncharacterized protein, WcoO of 272 aas and 8 TMSs.


WcoO of Clavibacter michiganensis


Uncharacterized protein of 155 aas and 5 TMSs.

UP of Methanosarcina mazei


Uncharacterized protein of 258 aas and 7 TMSs


UP of Bacillus selenitireducens


Uncharacterized protein of 290 aas and 8 TMSs


UP of Coriobacterium glomerans


Uncharacterized protein of 281 aas and 8 TMSs.


UP of Phytophthora infestans (Potato late blight fungus)


Uncharacterized protein of 264 aas and 8 TMSs.


UP of Rhizophagus irregularis (Arbuscular mycorrhizal fungus) (Glomus intraradices)


Uncharacterized protein of 350 aas and 8 TMSs.


UP of Guillardia theta


Uncharacterized protein of 277 aas and 7 TMSs

UP of Microbacterium yannicii


Uncharacterized protein of 236 aas and 8 TMSs in a 1 + 1 + 2 + 2 + 2 TMS arrangement.

UP of Paracoccus zeaxanthinifaciens


Uncharacterized protein of 250 aas and 8 TMSs.

UP of Henriciella aquimarina