Recombinant Candida glabrata Cytosolic Fe-S cluster assembly factor NAR1 (NAR1), partial

What is the basic structure and function of Candida glabrata NAR1?

C. glabrata NAR1 encodes a hydrogenase-like protein that functions as an essential component of the cytosolic iron-sulfur assembly (CIA) machinery. Structurally, NAR1 contains two conserved binding sites for [4Fe-4S] clusters. One site is predicted to be more stable, while the other functions as a labile site potentially involved in cluster transfer to downstream target proteins . Recent spectroscopic analyses have revealed that beyond the two [4Fe-4S] clusters, NAR1 can also unexpectedly bind a [2Fe-2S] cluster at an as-yet unidentified site . This structural arrangement indicates its specialized role in Fe/S protein biogenesis in the cytosol.

How does NAR1 differ from its homologs in other organisms?

NAR1 belongs to the Nar1 protein family found in virtually all eukaryotes, with members showing striking sequence similarity to bacterial iron-only hydrogenases, though they don't function as hydrogenases . The yeast homolog (Nar1p) is predominantly localized in the cytosol with some membrane association, which differs from its human homologue Narf that is exclusively nuclear . Functionality also varies significantly - human homologues (Narf and Hprn) cannot functionally replace Nar1p in yeast, suggesting evolutionary specialization despite structural similarities . The Aspergillus niger homolog shares the core function of cytosolic Fe-S cluster assembly but may exhibit species-specific regulatory mechanisms .

What is the subcellular localization of NAR1 in Candida glabrata?

Based on studies of the homologous protein in Saccharomyces cerevisiae, NAR1 is predominantly localized in the cytosol (approximately 80% of total protein) with a portion associated with microsomal membranes . Subcellular fractionation experiments have shown that NAR1 is effectively depleted in purified mitochondria, confirming it is not significantly associated with these organelles . This localization pattern is consistent with its role in cytosolic and nuclear Fe/S protein maturation rather than in mitochondrial Fe/S protein assembly.

What are the optimal expression systems for recombinant C. glabrata NAR1?

For successful expression of functional NAR1, E. coli-based systems have proven effective when optimized for Fe/S protein production . The expression protocol should include:

• Use of an E. coli strain with robust iron-sulfur cluster assembly machinery

• Growth under microaerobic or anaerobic conditions to prevent oxygen-mediated degradation of Fe/S clusters

• Supplementation with iron sources (typically ferric ammonium citrate) and sulfur sources

• Induction at lower temperatures (16-18°C) to promote proper protein folding

• Addition of cluster-stabilizing agents such as dithiothreitol (DTT) or β-mercaptoethanol during cell growth

Studies have shown that expression using the pET vector system with an N-terminal affinity tag (His or Strep) facilitates purification while preserving protein function . For C. glabrata-specific expression, the PDC1 promoter has been successfully employed as a constitutive promoter to replace GAL1, which provides only low expression levels in C. glabrata .

What purification strategies preserve the Fe/S clusters in recombinant NAR1?

Preserving the integrity of the Fe/S clusters during NAR1 purification requires specific techniques:

• Perform all purification steps under strict anaerobic conditions (typically in an anaerobic chamber with <1 ppm O₂)

• Use buffers degassed and supplemented with reducing agents (2-5 mM DTT or dithionite)

• Include glycerol (10-15%) to stabilize protein structure

• Maintain cold temperatures (4°C) throughout the purification process

• Use rapid purification protocols to minimize cluster degradation

For affinity chromatography, Strep-Tactin affinity chromatography has been successfully employed for purification of Strep-tagged Nar1p . Following initial purification, size exclusion chromatography can be used as a polishing step to separate fully assembled holoprotein from apoprotein forms .

How can the Fe/S clusters in recombinant NAR1 be characterized spectroscopically?

Multiple complementary spectroscopic techniques are required for comprehensive characterization of NAR1 Fe/S clusters:

Recent studies have revealed that recombinant NAR1 contains not only the expected [4Fe-4S] cluster but also an unexpected [2Fe-2S] cluster, detectable through combined spectroscopic approaches . The [4Fe-4S] clusters can be identified by a broad, unstructured absorption "shoulder" around 420 nm in UV-visible spectroscopy, which partially bleaches upon reduction with dithionite .

What spectroscopic features distinguish the different Fe/S clusters in NAR1?

The different Fe/S clusters in NAR1 can be distinguished through their unique spectroscopic signatures:

• The [4Fe-4S] cluster typically exhibits:

  • UV-Vis: Broad, unstructured absorption around 420 nm

  • EPR: Rhombic signal in the reduced state (g-values approximately 2.04, 1.94, 1.89)

  • Mössbauer: Characteristic quadrupole doublets

• The [2Fe-2S] cluster typically shows:

  • UV-Vis: More structured absorption features

  • EPR: Distinctive rhombic signal with g-values around 2.00, 1.96, 1.92

  • Mössbauer: Parameters distinct from [4Fe-4S] clusters

One key observation is that one of the [4Fe-4S] clusters in NAR1 is rapidly destroyed by molecular oxygen, potentially linking NAR1 oxygen sensitivity to phenotypes observed in vivo . This oxygen-labile cluster likely corresponds to the cluster at site 2, which is predicted to be involved in cluster transfer to target proteins.

How can NAR1 cluster transfer activity be assayed in vitro?

Assessing NAR1's function in Fe/S cluster transfer requires specialized in vitro assays:

• Direct Transfer Assays: Monitor transfer of Fe/S clusters from NAR1 to apo-acceptor proteins using:

  • UV-Visible spectroscopy changes during incubation of NAR1 with acceptor proteins

  • EPR spectroscopy to track changes in cluster environment

  • Activity restoration in Fe/S-dependent enzymes (e.g., aconitase)

• Coupled Enzyme Assays: Measure the activity of Fe/S-dependent enzymes following incubation with NAR1:

  • Establish baseline activity of apo-enzyme

  • Incubate with NAR1 (holo-form)

  • Measure restored enzymatic activity as indicator of successful Fe/S transfer

• In Vitro Reconstitution Systems: Reconstitute the entire CIA machinery by incorporating:

  • Purified CIA components (Cfd1, Nbp35, NAR1, Cia1, etc.)

  • Scaffold proteins

  • Target apo-proteins

  • Measure formation of holo-target proteins through activity or spectroscopic changes

Recent 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 facilitate the insertion of [4Fe-4S] and potentially [2Fe-2S] clusters into target proteins in the cytosol .

What is the relationship between NAR1 and azole resistance in C. glabrata?

While NAR1 itself is not directly implicated in azole resistance, it functions within the broader cellular context that influences antifungal susceptibility in C. glabrata. The prevalence of antifungal resistance in C. glabrata, especially against azole drugs, results in difficult-to-treat and potentially life-threatening infections .

Research has identified transcription factors like CgMar1 (Multiple Azole Resistance 1) that regulate numerous genes under fluconazole stress, including several related to lipid biosynthesis pathways . Many Fe/S proteins are involved in lipid metabolism and membrane composition, which affects drug permeability and accumulation.

Given that NAR1 is essential for the maturation of cytosolic and nuclear Fe/S proteins, it indirectly impacts cellular processes that contribute to drug resistance. Understanding NAR1's role in Fe/S protein maturation may provide insights into novel approaches for overcoming antifungal resistance.

How does oxygen sensitivity of NAR1 impact experimental design?

The oxygen sensitivity of NAR1's Fe/S clusters presents significant experimental challenges that must be addressed through careful experimental design:

• Anaerobic Handling Requirements:

  • All biochemical work must be conducted in anaerobic chambers (<1 ppm O₂)

  • Specialized equipment for spectroscopic analysis under anaerobic conditions

  • Sealed cuvettes for transfer between anaerobic chamber and instruments

• Experimental Implications:

  • Control experiments must assess the impact of unavoidable oxygen exposure

  • Time-course analyses to determine cluster degradation rates under various conditions

  • Comparison between wild-type NAR1 and oxygen-resistant mutants (if available)

• Physiological Relevance:

  • Design experiments to test how oxygen sensitivity relates to C. glabrata adaptation to various host niches

  • Investigate how NAR1 function persists in microaerobic environments

  • Explore potential regulatory mechanisms that might protect NAR1 function in vivo

Recent studies have demonstrated that one [4Fe-4S] cluster in NAR1 is rapidly destroyed by molecular oxygen, potentially linking its oxygen sensitivity to phenotypes observed previously in vivo . This sensitivity suggests that NAR1 may function as an oxygen sensor in addition to its role in Fe/S protein maturation.

What genetic approaches can be used to study NAR1 function in C. glabrata?

Given that NAR1 is essential for viability in yeast, specialized genetic approaches are required:

• Regulated Expression Systems:

  • Replace the native promoter with regulatable promoters (e.g., GAL1-10 or tetracycline-responsive elements)

  • Use the constitutive PDC1 C. glabrata promoter for controlled expression, as the GAL1 promoter allows only very low expression of downstream genes in C. glabrata

  • Create shutoff strains where NAR1 expression can be repressed to study depletion effects

• Domain-Specific Mutations:

  • Target conserved cysteine residues involved in Fe/S coordination

  • Introduce mutations in the two distinct Fe/S binding sites to differentiate their functions

  • Generate chimeric proteins with domains from NAR1 homologs to identify functional regions

• Complementation Studies:

  • Test whether NAR1 homologs from other species can complement C. glabrata NAR1 function

  • Previous studies with yeast showed that human homologues (Narf and Hprn) cannot functionally replace Nar1p, highlighting evolutionary specialization

• Genomic Approaches:

  • RNA-seq to identify genes differentially expressed upon NAR1 depletion

  • ChIP-seq to identify proteins that interact with NAR1 or are affected by its depletion

  • Comparative genomics across C. glabrata clinical isolates to identify potential variations in NAR1 and related pathways

How does NAR1 integrate with the broader Fe/S protein biogenesis machinery?

Understanding NAR1's role requires considering its position within the complex Fe/S protein biogenesis network:

• Dependence on Mitochondrial ISC Machinery:

  • The insertion of Fe/S clusters into NAR1 requires components of the mitochondrial iron-sulfur cluster (ISC) assembly machinery, including Nfs1p, Yah1p, and the ISC export machinery (Atm1p)

  • Depletion of these mitochondrial components leads to diminished Fe/S incorporation into NAR1 and eventually to degradation of NAR1 apoprotein

• Interaction with CIA Components:

  • NAR1 functions downstream of the CFD1-NBP35 scaffold complex that receives an unknown sulfur-containing compound exported from mitochondria

  • NAR1 potentially interacts with CIA1, CIA2, and MMS19 to form the CIA targeting complex

  • A critical research question is whether NAR1 directly receives clusters from the CFD1-NBP35 complex and transfers them to the CIA targeting complex

• Target Specificity:

  • NAR1 appears critical for maturation of both cytosolic and nuclear Fe/S proteins, but not mitochondrial ones

  • The mechanisms determining target specificity remain poorly understood

  • Current models suggest NAR1 may function as an adapter between the early and late steps of cytosolic Fe/S protein assembly

The relationship between NAR1 and the only other well-characterized component of the cytosolic pathway, the P-loop ATPase Cfd1p, remains an active area of investigation . Future research should address whether these proteins interact genetically or directly through protein-protein association.