Thousands of researchers around the world are studying the connections between mitochondria, metabolism and disease. MitoNews summarizes a selection of the latest published findings and highlights how Abcam's MitoSciences range of research tools has contributed to this effort. Read the full list of 28 original research papers published this month.
Past issues are available for review in the archives.
Mitochondrial calcium import
Calcium, in its ionic form (Ca2+), is a ubiquitous second messenger of eukaryotic cells, participating in numerous signal transduction pathways. The ER is the principal store of Ca2+ and mitochondria are important regulators of free Ca2+ by acting as a sink. High concentrations of Ca2+ have been demonstrated at the contact sites between the ER and mitochondria, therefore mitochondrial Ca2+ uptake must be regulated and rapid. Mitochondrial uptake of Ca2+ is membrane potential dependant and stimulates ATP synthesis by activating the rate limiting TCA cycle dehydrogenases PDH, ICDH and alpha KGDH. However Ca2+ overloading in mitochondria can also lead to the generation of reactive oxygen species, the opening of the permeability transition pore, cytochrome c release and apoptosis.
Ca2+ is stored in the mitochondrial matrix as a Ca-phosphate complex and is extruded through a Na+/Ca2+ exchanger or by the transient opening of the permeability transition pore (flickering). Until recently, the mechanism of Ca2+ entry into mitochondria was less clear. Ca2+ is accumulated via a ruthenium red sensitive, high capacity, uniporter termed the mitochondrial Ca2+ uniporter (MCU) and driven by the mitochondrial membrane potential but the molecular identity of this uniporter was unknown.
In 2010, Mootha and colleagues identified a key regulator of Ca2+ uptake termed MICU1 and, in 2011, the uniporter itself, MCU. MCU (previously CCDC109A) was identified by phylogenetics, and by RNA and protein co-expression profiling. These researchers went on to show a physical interaction between the GFP-tagged MCU and V5-tagged MICU1. Silencing MCU attenuated mitochondrial Ca2+ uptake, but respiration and membrane potential were unaffected. To its identify its sub-organellar localization, these researchers isolated mitochondria and showed that MCU was located within the inner mitochondrial membrane with matrix facing N and C termini by proteinase K protection assay using antibodies whose targets included CYCS, COX-II, and HSP60. Blue native separation revealed that MCU migrates within a complex at an apparent molecular weight of 450 kDa.
Mutation of four conserved acidic residues in the intermembrane space facing hairpin loop identified that three were critical to function and one, S259A, while not critical to function, confers marked resistance to an inhibitor, the Ruthenium red related compound Ru360. These studies indicate that the loop region is critical to function and inhibitor sensitivity.
Furthermore, the Rizzuto lab showed concurrently that MCU alone is able to form a Ca2+ pore. MCU protein was over expressed in both E.coli and wheat-germ expression systems followed by reconstitution into planar lipid bilayer, which formed a channel with the electrophysiological properties and inhibitor sensitivities of the Ca2+ uniporter. Rizzuto et al also showed, using a different proteinase protection assay, that MCU was a mitochondrial inner membrane protein. However, they proposed an opposite topology with N and C termini in the intermembrane space.
MICU1 encodes a mitochondrial EF hand protein required for (Ca2+) uptake.Nature 2010. Perocchi F, Gohil VM, Girgis HS, Bao XR, McCombs JE, Palmer AE, Mootha VK.
Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 2011. Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, Sancak Y, Bao XR, Strittmatter L, Goldberger O, Bogorad RL, Koteliansky V, Mootha VK.
A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 2011. De Stefani D, Raffaello A, Teardo E, Szabò I, Rizzuto R.
This month Mallilankaraman identified another Ca2+ uniporter complex associated protein. Using a directed RNAi screen of 45 mitochondrial membrane proteins, one protein, now termed MCU regulator 1 - MCUR1 (previously known as CCDC90A), markedly inhibited mitochondrial Ca2+ uptake in Hek293 cells, an effect reproduced in primary fibroblasts. Mitochondrial membrane potential was not affected, nor was MCU localization. Using classical outer membrane (VDAC) and inner membrane (COX-II) proteins as reference markers in a proteinase protection assay, they show that MCUR1 exists in the inner mitochondrial membrane and propose that it contains two transmembrane with the N and C termini facing the intermembrane space. Also using an immunoprecipitation strategy they showed that MCUR1 interacts with MCU but not MICU1, suggesting that MCU exists in a complex with MICU1 or MCUR1 but not both simultaneously.
MCUR1 is an essential component of mitochondrial (Ca2+) uptake that regulates cellular metabolism. Nature Cell Biol 2012 Mallilankaraman K, Cárdenas C, Doonan PJ, Chandramoorthy HC, Irrinki KM, Golenár T, Csordás G, Madireddi P, Yang J, Müller M, Miller R, Kolesar JE, Molgó J, Kaufman B, Hajnóczky G, Foskett JK, Madesh M.