Mechanistic and structural analysis of the function of the mitochondrial ABC transporter Atm1 in cellular iron-sulfur and iron metabolism

• Applicants: Professor Dr. Roland Lill; Vasundara Srinivasan, Ph.D.
• Subject Area: Biochemistry
• Term: from 2015 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 271743333

Mitochondria perform an essential function in the biogenesis of cellular iron-sulfur (Fe/S) proteins. These proteins are located in mitochondria, cytosol and nucleus, and fulfill important tasks in respiration, metabolism, protein translation, DNA synthesis and DNA repair. Their Fe/S co-factors are assembled and inserted into apoproteins by complex machinery. The mitochondrial iron-sulfur cluster (ISC) assembly machinery matures intra-organellar Fe/S proteins, yet is also indispensable for cytosolic and nuclear Fe/S protein assembly. It synthesizes a sulfur- and glutathione-containing factor termed X-S that is exported by the mitochondrial ISC export apparatus to the cytosol where X-S is used by early acting components of the CIA (cytosolic iron-sulfur protein assembly) machinery for maturation of extra-mitochondrial Fe/S proteins. The central component of the ISC export system is the mitochondrial ABC transporter Atm1. Its functional deficiency is associated with defects in cytosolic-nuclear Fe/S proteins and in cellular iron regulation. Mutations in its human ortholog ABCB7 cause the iron-storage disease X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). We recently solved the crystal structure of nucleotide-free Atm1 with and without bound glutathione. Despite its utmost physiological importance, precise molecular aspects of the ISC export reaction and Atm1 function are still poorly understood. Hence, the central goal of the current proposal is the mechanistic elucidation of the ISC export reaction and of Atm1 function. We want to functionally reconstitute the export process with isolated mitochondria and cytosolic [2Fe-2S] proteins or early-acting CIA components that according to our recent studies may function as acceptors of X-S. This system will be used to isolate and characterize X-S. We further want to expand the knowledge of how cytosolic [2Fe-2S] proteins are matured to better characterize the early part of the CIA machinery. Nothing is known so far about this branch of cytosolic Fe/S protein biogenesis. Further structural and mutational studies will be performed to unravel the transport cycle of Atm1. The investigations aim at the precise definition of the substrate binding pocket, the export channel within the membrane part of Atm1, and of the functional implication of disease-relevant mutations. Atm1 structures with bound substrate and/or different nucleotides may identify further snapshots of its transport cycle. Overall, the project aims to provide a mechanistic and structural view of the ISC export process and the role of Atm1.

• DFG Programme: Research Grants