Introduction:
Emerging evidence suggests that many RNA molecules currently annotated as noncoding contain short open reading frames that code for functional small proteins called microproteins. Microproteins play critical roles in a diverse range of essential biological processes. To identify novel cardiac-expressed microproteins, we used a comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid transmembrane protein that is abundantly expressed in the heart.
Methods and Results:
Subcellular localization studies showed that Mtlbn localized specifically to the inner mitochondrial membrane. Immunoprecipitations and mass spectrometry analysis indicated that Mtlbn interacted with subunits of complex III of the electron transport chain (ETC) and we observed the presence of Mtlbn in complex III respiratory supercomplexes (SCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). Cardiac-specific Mtlbn overexpressing transgenic (TG) mice and Mtlbn gene-deleted (knockout, KO) mice were generated to dissect the molecular function of this protein in the heart. Mtlbn TG mice developed cardiomyopathy and died prematurely with histological, biochemical and ultrastructural pathologic features. Metabolomic analysis indicated that hearts from TG mice had signs of increased oxidative stress and mitochondrial dysfunction. While Mtlbn KO mice had normal cardiac function and histological appearance, BN-PAGE analysis of purified mitochondria from KO hearts showed altered complex III composition. Functional assessment of purified heart mitochondria from Mtlbn KO mice revealed a reduction in complex III activity and metabolomic analysis of KO heart tissue indicated an altered metabolite profile consistent with deficiencies in complex III activity.
Conclusions:
Mtlbn is a novel heart-enriched microprotein that localizes to the inner mitochondrial membrane where it interacts with complex III of the ETC. Our data indicate that Mtlbn serves as a critical regulator of mitochondrial ETC activity through a direct role in affecting complex III function.