Role of the mitochondrial Bol1 and Bol3 proteins in iron-sulfur cluster delivery to diverse recipient proteins

• Applicant: Professor Dr. Roland Lill
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311061741

The biogenesis of mitochondrial iron-sulfur (Fe/S) proteins is catalyzed by the conserved ISC assembly machinery and can be dissected into three major steps. First, a [2Fe-2S] cluster is synthesized de novo on the scaffold protein Isu1 by the sulfur donor Nfs1-Isd11-Acp1 and four other ISC factors. Second, the Fe/S cluster is released from Isu1 by a dedicated Hsp70 chaperone system, and transferred via the monothiol glutaredoxin Grx5 to [2Fe-2S] target proteins. Third, a transiently Grx5-bound [2Fe-2S] cluster is converted by the Isa1-Isa2-Iba57 proteins to a [4Fe-4S] cluster, which is transiently bound by the specific ISC targeting factors Nfu1 and Ind1, and finally inserted into recipient [4Fe-4S] proteins. Work within this project has identified the two yeast mitochondrial proteins Bol1 and Bol3 as additional ISC targeting factors that cooperatively facilitate specific maturation of lipoyl synthase and respiratory complex II. Human BOLA3 (Bol3 homolog) but not BOLA1 performs a similar targeting function. The solution structure of the two BOLA proteins was resolved in cooperation with L. Banci. Biochemical studies revealed rather different affinities of the two BOLA complexes with [2Fe-2S] cluster-bound GLRX5 (human Grx5 homolog), yet the physiological meaning of this observation remains unclear. Mutational analysis indicated that the [2Fe-2S] cluster is bound to the BOLA1-GLRX5 complex independently of three conserved His residues in BOLA1, leaving the exact architecture of the holo-complex open. Our work has raised the central question of why a [2Fe-2S] cluster-containing Bol-Grx5 complex is needed for [4Fe-4S] protein assembly. Further, based on the radically different BOL and GRX5 deletion phenotypes in both yeast and human cells (and in diseases), it became evident that the precise site of action of Bol-Grx5 within the complex Fe/S protein assembly pathway remains open. In the next funding period, we aim to reach a physiologically meaningful mechanistic understanding of Bol protein function that explains the involvement in [4Fe-4S] cluster insertion into mitochondrial apoproteins. We will test whether the Bol proteins exhibit an increased importance under oxidative stress conditions by examining their function and interacting partner proteins. We will reconstitute lipoyl synthase maturation in vitro to precisely define the mechanistic function of the Bol and Grx5 proteins. In support of this aim, the crystal structure of the Bol-Grx5 complexes will be solved. Finally, we will perform cell biological and biochemical studies to elucidate whether the Bol-Grx5 complexes act before or after the Isa-Iba57 proteins. A comprehensive mechanistic understanding of Bol protein function will be important for a molecular view of the BOLA3-associated mitochondrial disease MMDS2.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life