Biology subjects

Braymer, J. J.

Publications and source records attributed to Braymer, J. J..

3 recordsLinked to original sources

bioRxiv2025

Yeast cluster

Nar1 is an essential eukaryotic protein proposed to function as an iron-sulfur (Fe/S) cluster trafficking factor in the cytosolic iron-sulfur assembly (CIA) machinery. However, such a role has remained unclear due to difficulties in purifying adequate amounts of cofactor-bound protein. The [FeFe]-hydrogenase-like protein has two conserved binding sites for [4Fe-4S] clusters, one of which is predicted to be a labile site for cluster transfer to downstream targets. Here, we report a new preparation procedure for Nar1 that facilitated studies by UV-Vis, EPR, and Mossbauer spectroscopies, along with native mass spectrometry. Nar1 recombinantly produced in E. coli contained a [4Fe-4S] cluster, bound presumably at site 1, along with an unexpected [2Fe-2S] cluster bound at an unknown site. Fe/S reconstitution reactions installed a second [4Fe-4S] cluster at site 2, leading to protein with three Fe/S cofactors. Strikingly, one [4Fe-4S] cluster was rapidly destroyed by molecular oxygen, potentially linking Nar1 oxygen sensitivity to phenotypes observed previously in vivo. These advances now allow for the pursuit of in vitro Fe/S cluster transfer assays, which will shed light on Fe/S trafficking by CIA components and how they may facilitate the insertion of [4Fe-4S] and potentially [2Fe-2S] clusters into target proteins in the cytosol.

biochemistry↗

bioRxiv2024

Requirements for the Biogenesis of Proteinsin the Human and Yeast Cytosol

The biogenesis of iron-sulfur (Fe/S) proteins entails the synthesis and trafficking of Fe/S clusters, followed by their insertion into target apoproteins. In eukaryotes, the multiple steps of biogenesis are accomplished by complex protein machineries in both mitochondria (ISC) and cytosol (CIA). The underlying biochemical pathways have been elucidated over the past decades, yet the mechanisms of cytosolic [2Fe-2S] protein assembly have remained ill-defined. Similarly, the precise site of glutathione (GSH) requirement in cytosolic and nuclear Fe/S protein biogenesis is unclear, as is the molecular role of the GSH-dependent cytosolic monothiol glutaredoxins (cGrxs). Here, we investigated these questions in human and yeast cells by various in vivo approaches. [2Fe-2S] cluster assembly of cytosolic target apoproteins required the mitochondrial ISC machinery, the ABC transporter Atm1/ABCB7 and GSH, yet occurred independently of both the CIA system and cGrxs. This mechanism was strikingly different from the ISC-, Atm1/ABCB7-, GSH-, and CIA-dependent assembly of cytosolic-nuclear [4Fe-4S] proteins. One notable exception to this newly defined cytosolic [2Fe-2S] protein maturation pathway was the yeast protein Apd1 which used the CIA system via binding to the CIA targeting complex through its C-terminal tryptophan. cGrxs, although attributed as [2Fe-2S] cluster chaperones or trafficking proteins, were not essential in vivo for deliver ing [2Fe-2S] clusters to either CIA components or apoproteins. Finally, GSH function was assigned to Atm1-dependent export, i.e. a step before GSH-dependent cGrxs function. Our findings extend the general model of eukaryotic Fe/S protein biogenesis by adding the molecular requirements for cytosolic [2Fe-2S] protein maturation.

biochemistry↗

bioRxiv2023

Characterisation of the SUF FeS cluster machinery in the amitochondriate eukaryote Monocercomonoides exilis

Monocercomonoides exilis is the first eukaryotic organism described as a complete amitochondriate, yet it shares common features with heterotrophic anaerobic/microaerophilic protists, some of which bear divergent mitochondrion-related organelles or MROs. It has been postulated that the retention of these organelles stems from their involvement in the assembly of essential cytosolic and nuclear FeS proteins, whose maturation requires the evolutionarily conserved mitochondrial ISC and cytosolic CIA machineries. The amitochondriate M. exilis lacks genes encoding the ISC machinery yet contains a bacteria-derived SUF system (MeSuf), composed of the cysteine desulphurase SufS fused to SufD and SufU, as well as the FeS scaffolding components MeSufB and MeSufC. Here, we show that expression of the M. exilis SUF genes, either individually or in tandem, can restore the maturation of the FeS protein IscR in the Escherichia coli double mutants of {Delta}sufS {Delta}iscS and {Delta}sufB {Delta}iscUA. In vivo and in vitro studies indicate that purified MeSufB, MeSufC and MeSufDSU proteins interact suggesting that they act as a complex in the protist. MeSufBC can undergo conformational changes in the presence of ATP and assemble FeS clusters under anaerobic conditions in presence and absence of ATP in vitro. Altogether, these results indicate that the dynamically interacting MeSufDSUBC proteins may function as an FeS cluster assembly complex in M. exilis thereby being capable of replacing the organelle-enclosed ISC system of canonical eukaryotes.

biochemistry↗