BOLA1

BOLA1 is a mitochondrial protein that buffers the mitochondrial thiol redox potential against glutathione depletion, with knockdown increasing oxidation of mitochondrial thiols and overexpression protecting mitochondrial morphology from oxidative challenge (PMID:22746225). It executes this function through a direct physical partnership with the monothiol glutaredoxin GLRX5 (PMID:22746225), forming a [2Fe-2S] cluster-bridged heterodimer that uniquely coordinates a reduced, Rieske-type [2Fe-2S]1+ cluster and is preferentially assembled over the related BOLA3-GLRX5 complex, which instead holds an oxidized ferredoxin-like cluster (PMID:28483642). In vitro, this heterodimer can acquire its cluster from ISCU or glutathione-coordinated [2Fe-2S] but not from ISCA1/ISCA2, and once formed cannot donate the cluster to apo acceptors, marking BOLA1-GLRX5 as a redox-active rather than iron-sulfur trafficking species (PMID:32542995). Consistent with this non-essential role in cluster biogenesis, knockout of the BolA1 homologue in Giardia mitosomes leaves [2Fe-2S] cluster formation intact (PMID:37792908).

1. 2012 Medium

   Established that BOLA1 is a mitochondrial factor controlling the thiol redox environment, answering whether the uncharacterized protein had any defined cellular function.

   Evidence Subcellular fractionation, siRNA knockdown and overexpression with BSO/S-nitrosocysteine, plus mitochondrial thiol redox potential measurement in cultured cells

   PMID:22746225

   Open questions at the time

   • Molecular mechanism linking BOLA1 to thiol potential not defined
   • Did not identify the cofactor or binding partner mediating the effect
2. 2012 Medium

   Identified GLRX5 as a direct BOLA1 partner, providing the molecular link between BOLA1 and glutaredoxin-based redox chemistry.

   Evidence Co-immunoprecipitation

   PMID:22746225

   Open questions at the time

   • Single Co-IP without reciprocal or stoichiometric validation at this stage
   • Nature of the interaction interface and any shared cofactor unknown
3. 2014 Medium

   Resolved the structural basis of BolA-glutaredoxin assembly and cluster coordination using the Arabidopsis ortholog, revealing a Rieske-type [2Fe-2S] cluster ligated by a variable-loop histidine and a defined heterodimer interface.

   Evidence X-ray crystallography of three BolA structures, 3D modeling, spectroscopy and NMR interaction experiments on the plant ortholog

   PMID:25012657

   Open questions at the time

   • Plant ortholog, not human BOLA1
   • Functional consequence of the Rieske-type coordination not established in this study
4. 2017 High

   Defined the human BOLA1-GLRX5 complex as a [2Fe-2S]-bridged heterodimer holding a reduced Rieske-type cluster, distinguishing it from the oxidized cluster of BOLA3-GLRX5 and establishing a redox-distinct identity.

   Evidence UV/vis, CD, EPR and NMR spectroscopy with experimentally-driven protein-protein docking

   PMID:28483642

   Open questions at the time

   • In-cell relevance of the reduced cluster redox state not demonstrated
   • Physiological electron donor/acceptor of the cluster unidentified
5. 2020 Medium

   Determined the directionality of cluster exchange, showing the heterodimer receives clusters from ISCU or glutathione-coordinated [2Fe-2S] but cannot donate them, ruling against a trafficking role and supporting a redox function.

   Evidence CD spectroscopy-based in vitro kinetic cluster exchange assays with defined donors and acceptors

   PMID:32542995

   Open questions at the time

   • Single in vitro method, single lab
   • Physiological redox substrate not identified
   • Relevance of the reactive BOLA1 homodimer in cells unknown
6. 2023 Medium

   Tested whether BOLA1 is required for iron-sulfur cluster biogenesis, finding it dispensable for core [2Fe-2S] formation in a divergent anaerobic organism while confirming the conserved Grx5 interaction.

   Evidence Affinity pulldown, CRISPR/Cas9 knockout and in vitro [2Fe-2S] cluster formation assay in Giardia intestinalis mitosomes

   PMID:37792908

   Open questions at the time

   • Divergent protist ortholog may not reflect human biology
   • Redox-buffering role not directly tested by this knockout

• The physiological electron donor/acceptor of the reduced BOLA1-GLRX5 cluster and the in-cell mechanism by which BOLA1 buffers the mitochondrial thiol potential remain unresolved.
• No identified physiological redox partner for the cluster
• No structural model of the human holo-complex from direct experimental coordinates
• Link between cluster redox state and the measured thiol-potential phenotype not mechanistically closed