Molecular determinants for cytosolic iron-sulfur cluster insertion

• Applicant: Professor Dr. Antonio J. Pierik
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology ; Biophysics
• Term: from 2016 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311771359

During the last few years a significant increase in the understanding of cytosolic and nuclear iron sulfur (FeS) protein maturation has been achieved. However, for most eukaryotic FeS target proteins few or no mechanistic details for cluster insertion have been described. In contrast to mitochondrial FeS proteins, 20 % of the yeast and 14 % of the human cytosolic and nuclear FeS proteins have a tryptophan residue at their C-terminus, commonly occurring in the L(D/E)W tripeptide. Our in vivo data in Saccharomyces cerevisiae (yeast) and in vitro results within the first funding period demonstrated that the FeS cluster insertion by the CIA machinery, the function of the CIA-component Nar1, the isopropylmalate isomerase Leu1 and the DNA polymerase Pol3 critically depend on the tryptophan residue at their C-terminus. For Pol3 the tryptophan residue was critical only under DNA-damaging conditions, suggesting that the [4Fe-4S] cluster might not be primarily related to DNA replication, but to repair. Leu1 served as a excellent model to test loss and gain of function. Using variants of yeast Leu1 and engineered E. coli isopropylmalate isomerase (LeuCD) fused to yeast Leu1 C-terminal determinants, we could show that not only the tryptophan residue, but also an optimal length of the extension and the accessibility of the last residue are critical determinants. In subsequent experiments, the universality of our system will be tested using C-terminal determinants of phylogenetically diverse fungal Leu1 proteins and C-termini of other cytosolic Fe/S proteins (Nar1, Pol3, viperin) fused to Leu1/LeuCD system in yeast. We will develop new molecular toolboxes as alternative for the radioactive iron based maturation analysis using in vivo and cell extract cluster-type selective EPR spectroscopy and, employing stable iron isotopes, ICP-MS after pulldown. Our preliminary data demonstrate that very intense and specific EPR signals for Apd1, an intrinsic yeast cytosolic bis-histidinyl coordinated [2Fe-2S] protein, other [2Fe-2S] proteins from Fungi and several FeS dehydratases can be quantified in yeast cell extract. Using these approaches, we aim to dissect the CIA machinery into [2Fe-2S] and [4Fe-4S] specific segments at molecular level. Using the relatively thermo- and O2-stable 6-phosphogluconate and dihydroxyacid dehydratase enzymes we will explore the use of the C-terminal extension for biotechnological applications of non-yeast, FeS cluster-containing enzymes. The culmination will be our trial to engineer an active FeFe hydrogenase in yeast cytosol.

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