Distinct roles for the domains of the mitochondrial aspartate/glutamate carrier citrin in organellar localization and substrate transport
Sotiria Tavoulari, Denis Lacabanne, Gonçalo C. Pereira, Chancievan Thangaratnarajah, Martin King, Jiuya He, Suvagata Roy Chowdhury, Lisa Tilokani, Shane M. Palmer, Julien Prudent, John E. Walker, Edmund R.S. Kunji
Abstract
Citrin, the mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), is structurally and mechanistically the most complex SLC25 family member, because it consists of three domains and forms a homo-dimer. Each protomer has an N-terminal calcium-binding domain with EF-hands, followed by a substrate-transporting carrier domain and a C-terminal domain with an amphipathic helix. The absence or dysfunction of citrin leads to citrin deficiency, a highly prevalent pan-ethnic mitochondrial disease. Here, we aim to understand the role of different citrin domains and how they contribute to pathogenic mechanisms in citrin deficiency. We have employed structural modeling and functional reconstitution of purified proteins in proteoliposomes to assess the transport activity and calcium regulation of wild-type citrin and pathogenic variants associated with citrin deficiency. We have also developed a double knockout of citrin and aralar (AGC1), the two paralogs of the mitochondrial aspartate/glutamate carrier, in HAP1 cells to perform mitochondrial imaging and to investigate mitochondrial localisation. Using 33 pathogenic variants of citrin we clarify determinants of subcellular localization and transport mechanism. We identify crucial elements of the carrier domain that are required for transport, including those involved in substrate binding, network formation and dynamics. We show that the N-terminal domain is not involved in calcium regulation of transport, as previously thought, but when mutated causes a mitochondrial import defect. Our work introduces a new role for the N-terminal domain of citrin and demonstrates that dysfunction of the different domains contributes to distinct pathogenic mechanisms in citrin deficiency. • We study 33 citrin missense mutations causing citrin deficiency. • We identify elements crucial for transport within the carrier domain of citrin. • The transport activity of citrin is not calcium-regulated, as previously thought. • Pathogenic mutations in the N-terminal domain cause a mitochondrial import defect. • Citrin domains contribute to distinct pathogenic mechanisms in citrin deficiency.