Mitochondrial mayhem: Disrupting conserved N-terminal motifs in TANGO2 impacts its localization and function

Transport and Golgi organization 2 (TANGO2) is an ancient protein with high conservation across all domains of life. Nonetheless, research interest on TANGO2 has only surged in the last decade, following its linkage with a rare neurodegenerative disease (1, 2). Initially identified in an RNAi screen in Drosophila focused on Golgi structural integrity (3), more recent studies suggest that TANGO2 may be critical for mitochondrial function and lipid homeostasis (4, 5, 6). The clinical phenotype observed in TANGO2 deficiency disorder (TDD), an autosomal recessive loss-of-function condition, supports this notion, bearing similarity to that of several other diseases linked to coenzyme A (CoA) or acyl-CoA metabolism. At baseline, individuals with TDD may experience intellectual disability, impaired speech, seizures, movement disorders, and intermittent periods of extreme fatigue and decreased responsiveness. Fasting, fever, or other triggers of metabolic stress may precipitate metabolic crises, which are marked by lactic acidosis, hypoglycemia, rhabdomyolysis, and ventricular tachyarrhythmias. Pantothenic acid, an obligate precursor of CoA, appears to exert a therapeutic effect in patients with TDD, though the mechanism driving this benefit remains unknown at present (7).

In this issue of JCB, work by Lujan and colleagues offers new insights into the localization and function of TANGO2 (8). Building on prior studies showing variable localization to the cytosol and mitochondria, Lujan et al. used time-lapse confocal microscopy in Tom20-labeled Hep2G and U2OS cells to track mScarlet-tagged TANGO2 to the mitochondrial lumen. TANGO2 abundance was also measured following mitochondrial exposure to proteinase K with and without Triton-X, detergents that lyse the outer and inner mitochondrial membranes, respectively. Following treatment with proteinase K alone, a significant amount of TANGO2 was recovered and visualized via immunoblot, suggesting its presence in the lumen.

Lujan et al. also modified two conserved amino acid sequences at the N terminus of TANGO2 and showed that the LIL motif, but not a nearby NRDE motif, is required for mitochondrial localization (Fig. 1). Following mutagenesis of LIL to AGA, they observed that mutant TANGO2 predominantly localized to the cytosol at the periphery of lipid droplets. Leucine is a large, hydrophobic amino acid, and a pathogenic missense variant coding for a hydrophilic arginine residue at the more highly conserved first leucine position of LIL (c.59T>G and p.L20R) has been reported in individuals with TDD (4).

The aforementioned NRDE motif, just three amino acids away from the LIL sequence lines on one side of a putative hydrophobic binding pocket and is highly conserved. The presence of this pocket has been postulated since the advent of high-fidelity in silico protein modeling tools (10) and was recently confirmed via crystallography (PDB ID 8SV7). Lujan et al. first demonstrated that TANGO2 does not exhibit clear acyltransferase activity via protein overexpression, substrate exposure, and mass spectroscopy quantification of phospholipid byproducts. They then employed a series of fluorescence quenching assays to show that TANGO2 does, however, bind to 16- and 18-carbon acyl-CoA species as well as CoA itself. This binding capacity was essentially ablated following mutagenesis of the NRDE motif, underscoring its functional importance.

Missense variants affecting residues outside of the LIL and NRDE motifs have also been found in individuals with TDD. For example, a threonine to isoleucine change within the YGTR motif on the beta sheet strand adjacent to NRDE is known to be pathogenic (11). Presumably, this variant also results in impaired binding of hydrophobic ligands, though this has not been experimentally demonstrated.

While this work advances our understanding of the localization and function of TANGO2, several unanswered questions remain. Though it is tempting to infer that TANGO2 is involved in mitochondrial lipid trafficking, additional studies are needed to substantiate this hypothesis. Lujan et al. demonstrated that TANGO2 can move between the cytoplasm and mitochondria depending on cellular conditions, but it is not yet clear whether TANGO2 is actively shuttling molecules between these compartments. Second, more work is needed to determine the promiscuity of the hydrophobic binding pocket. For example, three recent papers have suggested that TANGO2 plays a role in heme transport (albeit via different mechanisms) (12, 13, 14), and heme itself is a highly hydrophobic molecule. It is also possible that TANGO2 primarily interacts with hydrophobic domains of other proteins or with membrane-bound lipids via its binding pocket. Third, although Lujan et al. demonstrated that TANGO2 does not have acyltransferase activity with respect to the specific substrates tested, other enzymatic functions cannot be excluded. The binding pocket may also indirectly mediate the function of TANGO2 via allosteric regulation. Finally, it is worth noting that TANGO2 homologs exist across a wide variety of species, including prokaryotes. The mitochondrial localization of TANGO2 in human cells and its potential shuttling between the cellular compartments raises questions as to its function in species that lack mitochondria.

While TDD affects thousands of individuals worldwide and is associated with high morbidity and mortality, ongoing exploration through both basic discovery and engagement with the patient community gives cause for hope. Disease models of TDD now span five different species, and multiple patient-derived cell lines have been established. To our knowledge, research is being conducted in at least seven countries. As with the impactful observations related to mitochondrial localization and acyl-CoA binding by Lujan and colleagues, each new insight related to the role of TANGO2 represents one step further along the path to an effective treatment for individuals with TDD. Additionally, although TDD is a rare disorder, efforts to understand the function of TANGO2 are likely to increase our broader understanding of mitochondrial biology and cellular metabolism.