Project Summary The human mitochondrial inner membrane has three ABC transporters: ABCB7 implicated in iron transport (with mutations causing X-linked sideroblastic anemia with ataxia), ABCB8 involved in regulation of a mitochondrial K+ channel and protection against doxorubicin toxicity, and ABCB10, which is essential for red blood cell development, protection against oxidative stress and whose complete absence leads to death of mice embryos. Despite their clear importance, little molecular research has been done on these human mitochondrial transporters, mainly because production of human membrane proteins is difficult. We believe that facilitating the production of these transporters will accelerate the research needed for a better molecular understanding of the mechanisms underlying their function. Human ABC transporters had only been produced in expensive and time-consuming eukaryotic systems until our group recently had the idea of producing ABCB10 in bacteria. Our preliminary studies show that ABCB10 produced in bacteria is functional. Here, we propose to use this novel approach for the easy, fast, and inexpensive production of ABCB7 and ABCB8. Aim 1 will develop methodologies for expression and purification of functional ABCB7 and ABCB8 in bacteria. Aim 2 will optimize methodologies for reconstitution of the purified transporters into a lipid bilayer (nanodiscs and liposomes) to perform functional assays. Our proposal is of high risk because we will test the feasibility of producing these human mitochondrial transporters in bacteria. This proposal is significant because it will provide optimized methodologies for production and functional testing of two important human proteins that have barely been studied, which will be of high reward. The experience of our group on ABC transporters molecular research, combined with these new methodologies, will provide a pathway for a more ambitious R01 proposal aimed at studying the fundamental molecular mechanisms of these human mitochondrial transporters.