Fig. 3: POI-causing AARS2 mutants gain lactyltransferase activities.

A–B R199C gained lactyltransferase activity. Lac-K336 levels (A) and Lac-K457/8 levels (B) of PDHA1-Flag and CPT2-Flag that were expressed from AARS2^−/−COV434, and from AARS2^−/− COV434 that co-expressed AARS2, N104Y, or R199C were detected. C–D R199C has stronger lactyltransferase activity than AARS2. The abilities to form Lac-K336 in purified PDHA1 (C) and Lac-K457/8 in purified CPT2 (D) of AARS2 and R199C were detected in vitro. E–F All POI-causing AARS2 mutants gained lactyltransferase activity. Lac-K336 (E) and Lac-K457/8 (F) levels were detected in immunoprecipitated PDHA1-Flag and CPT2-Flag purified from COV434, AARS2^−/− COV434, and AARS2^−/− COV434 cells ectopically expressing AARS2, F50C, T328K, or A77V. G POI-causing AARS2 mutations cluster around substrate-binding pocket. Disease-causing sites are marked in human AARS2 structure. POI-associated mutations are shown as spheres and labeled in red, cardiomyopathy-associated mutations are labeled in green, and leukodystrophy-associated mutations are labeled in violet. Aminoacylation subdomain (35–312 aa, gray) is in gray, tRNA recognition subdomain (313–477 aa) is in green, linker (478–529 aa) between tRNA recognition and editing domain is in blue, and editing domain (530–783 aa) and C-terminal domain (784-985) are in magenta and cyan, respectively. Backbone of docked tRNA and alanyl-adenylate are shown in orange and rainbow, respectively. H R199C has stronger binding ability for lactate. Isothermal Titration Calorimetry (ITC) was employed to measure the binding affinity of lactate to AARS2 (balk line) and R199C (red line). I–J R199C inhibits CPT2 and PDHA1 more strongly than AARS2. CPT2 (I) and PDC (J) specific activities were detected in purified CPT2 and cell lysates from COV434, AARS2^−/− COV434, and AARS2^−/− COV434 that co-expressed AARS2 or R199C, respectively (n = 3).