Recombinant Lagothrix lagotricha Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)

What is UQCRFS1 and what role does it play in mitochondrial function?

UQCRFS1 (Ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1) is a critical catalytic subunit of respiratory Complex III (cytochrome bc1 complex). It contains a 2Fe-2S cluster essential for electron transfer during oxidative phosphorylation. The protein functions within the mitochondrial respiratory chain as part of Complex III, which consists of products from one mitochondrially encoded gene (MTCYTB) and ten nuclear genes, including UQCRFS1 . As the Rieske iron-sulfur protein component, it catalyzes electron transfer from ubiquinol to cytochrome c, contributing to the proton gradient necessary for ATP synthesis. Its proper incorporation and function are essential for cellular respiration and energy production .

What is unique about Lagothrix lagotricha UQCRFS1 compared to other species?

The Lagothrix lagotricha (Brown woolly monkey or Humboldt's woolly monkey) UQCRFS1 protein represents an important primate model for studying mitochondrial function. The recombinant version encompasses amino acids 79-274, representing the mature form of the protein after removal of its mitochondrial targeting sequence . Comparative analysis places this protein in an evolutionary context between human and other primate UQCRFS1 variants. Recent phylogeographic studies of Lagothrix lagotricha have revealed complex evolutionary patterns, with well-defined evolutionary units that do not necessarily correlate with morphological classifications . This makes the woolly monkey UQCRFS1 particularly interesting for comparative mitochondrial studies, as it may provide insights into functional adaptations of respiratory complexes across primate evolution.

How is the UQCRFS1 protein processed in mitochondria?

The processing of UQCRFS1 follows a unique pathway unlike most mitochondrial proteins. UQCRFS1 is initially synthesized with a 78-amino acid N-terminal presequence that functions as a mitochondrial targeting signal . Upon import into mitochondria, this presequence is cleaved in a single proteolytic step after the protein's incorporation into the cytochrome bc1 complex . Fascinatingly, unlike most targeting presequences that are degraded after cleavage, the UQCRFS1 presequence remains as subunit 9 of the complex . This represents a rare dual-function peptide that serves first in protein trafficking and then as a structural component of the mature respiratory complex. The incorporation of UQCRFS1 requires the action of BCS1L, which translocates and integrates the protein with its 2Fe-2S cluster into the pre-CIII2 complex, activating its catalytic function . Additionally, the cleavage of the mitochondrial targeting sequence occurs only after incorporation into Complex III, not before .

What are the optimal conditions for handling recombinant Lagothrix lagotricha UQCRFS1?

The recombinant Lagothrix lagotricha UQCRFS1 protein with N-terminal His tag, expressed in E. coli, requires specific handling conditions to maintain stability and function. The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being optimal) and store in aliquots at -20°C/-80°C to prevent repeated freeze-thaw cycles which degrade protein quality . Working aliquots can be stored at 4°C for up to one week . The protein is buffered in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain stability during storage . Prior to opening, vials should be briefly centrifuged to bring contents to the bottom of the container. The reconstituted protein has greater than 90% purity as determined by SDS-PAGE, making it suitable for various biochemical and functional studies .

How can researchers assess UQCRFS1 protein expression and Complex III assembly?

Researchers can employ several complementary approaches to assess UQCRFS1 expression and Complex III assembly:

• Immunoblotting: SDS-PAGE followed by western blotting using antibodies against UQCRFS1 (such as Abcam ab14746, 1:1,000 dilution) can quantify UQCRFS1 protein levels . Controls should include antibodies against other mitochondrial proteins like SDHA (1:3,000, Abcam ab14715) and citrate synthase (1:3,000; THP, NBP2-43648) .

• Blue Native PAGE (BN-PAGE): This technique allows visualization of intact respiratory chain complexes and supercomplexes to assess Complex III assembly status.

• Mitochondrial Import Assays: In vitro transcription and translation of UQCRFS1 precursor, followed by incubation with isolated mitochondria, can assess proper import and processing . The processed product should comigrate with subunit 9 on SDS-PAGE .

• Enzyme Activity Assays: Complex III activity can be measured spectrophotometrically by monitoring the reduction of cytochrome c at 550 nm.

• Cellular Respiration: Oxygen consumption rates in cells expressing recombinant UQCRFS1 can be measured using instruments like Seahorse XF Analyzer to assess functional integration into the respiratory chain .

These methods collectively provide a comprehensive assessment of both UQCRFS1 protein expression and its functional incorporation into Complex III.

What experimental approaches can be used to study the 2Fe-2S cluster assembly in UQCRFS1?

Studying the 2Fe-2S cluster assembly in UQCRFS1 requires specialized techniques to track both protein maturation and iron-sulfur cluster incorporation:

• Co-immunoprecipitation Assays: These can identify interactions between UQCRFS1 and assembly factors like LYRM7 and HSC20, which are essential for 2Fe-2S cluster transfer and incorporation .

• EPR Spectroscopy: Electron paramagnetic resonance spectroscopy can directly detect the presence and integrity of the 2Fe-2S cluster in purified UQCRFS1 protein.

• Iron-55/Sulfur-35 Labeling: Radioactive labeling followed by immunoprecipitation can trace the assembly of new Fe-S clusters into UQCRFS1.

• Proximity Labeling Techniques: BioID or APEX2 fusion proteins can identify proximal proteins involved in Fe-S cluster insertion.

• Mutagenesis of Coordinating Residues: Site-directed mutagenesis of the cysteine residues that coordinate the Fe-S cluster can assess the importance of these sites for cluster binding and protein function.

The 2Fe-2S cluster incorporation requires direct binding of co-chaperone HSC20 to LYRM7, which recruits the 2Fe-2S transfer complex to the LYRM7-UQCRFS1 intermediate . This complex process ensures proper insertion of the critical iron-sulfur cluster that makes UQCRFS1 catalytically active.

How can researchers model UQCRFS1-related mitochondrial disorders in experimental systems?

Modeling UQCRFS1-related disorders requires multiple complementary approaches:

• Patient-Derived Fibroblasts: Primary fibroblasts from patients with UQCRFS1 mutations provide a direct cellular model to study pathophysiology. These cells typically show reduced UQCRFS1 protein levels, impaired CIII assembly, and decreased cellular respiration .

• CRISPR/Cas9 Gene Editing: Introducing patient-specific mutations into cell lines or model organisms can recreate disease phenotypes. Both homozygous and compound heterozygous mutations can be modeled to reflect the bi-allelic nature of UQCRFS1-related disorders .

• Lentiviral Complementation: Wild-type UQCRFS1 can be overexpressed via lentiviral transduction in patient cells to confirm causality and rescue cellular phenotypes. This approach has successfully restored mitochondrial function in cells with UQCRFS1 mutations .

• Inducible Knockdown Models: shRNA or siRNA against UQCRFS1 can create titratable models of protein deficiency.

• Transgenic Animal Models: While challenging due to potential embryonic lethality, conditional knockout or knock-in models can provide insights into tissue-specific effects of UQCRFS1 deficiency.

These models allow researchers to study the molecular consequences of UQCRFS1 deficiency, including effects on Complex III assembly, respiratory chain function, reactive oxygen species production, and mitochondrial dynamics. Clinical manifestations associated with UQCRFS1 mutations include lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis , which can be investigated using these experimental systems.

What methodological challenges exist in cross-species UQCRFS1 comparative studies?

Cross-species studies of UQCRFS1 face several methodological challenges:

The taxonomic complexity of Lagothrix lagotricha adds another layer of difficulty, as recent phylogeographic studies reveal evolutionary units that don't correlate well with morphological classifications . When using Lagothrix lagotricha UQCRFS1 in comparative studies, researchers should consider the precise origin of samples, as there is ongoing debate about raising subspecies to species level . The lack of reciprocal monophyly between putative subspecies (L. l. poeppiggi, L. l. lagotricha, and L. l. lugens) suggests ancestral polymorphism maintained during the spread of woolly monkeys throughout western Amazonian lowlands and into the inter-Andean region of Colombia .

How can researchers analyze the effects of UQCRFS1 mutations on protein stability and function?

Analyzing UQCRFS1 mutations requires a multi-parametric approach:

• In Silico Prediction: Computational tools can predict the impact of mutations on protein structure, stability, and function. These include SIFT, PolyPhen-2, and molecular dynamics simulations that can model effects on the 2Fe-2S cluster coordination.

• Thermal Stability Assays: Differential scanning fluorimetry can assess changes in protein stability caused by mutations. Decreased stability often correlates with functional defects and accelerated protein degradation.

• Pulse-Chase Experiments: Radioactive labeling followed by immunoprecipitation at different time points can measure protein half-life and degradation rates in cells expressing mutant UQCRFS1 compared to wild-type.

• Subcellular Fractionation: This technique can determine whether mutations affect mitochondrial targeting and import by quantifying the distribution of UQCRFS1 between cytosolic and mitochondrial fractions.

• BN-PAGE Analysis: Blue Native gel electrophoresis can visualize defects in Complex III assembly resulting from UQCRFS1 mutations .

• Enzymatic Activity Assays: Spectrophotometric methods measuring Complex III activity can quantify the functional impact of mutations.

• Complementation Studies: Expression of wild-type UQCRFS1 in cells carrying mutations can confirm pathogenicity if it rescues the phenotype .

These approaches collectively provide a comprehensive assessment of how mutations affect UQCRFS1 at the molecular and cellular levels, helping researchers understand the mechanistic basis of associated mitochondrial disorders.

What are the optimal expression systems for producing functional recombinant UQCRFS1?

The choice of expression system significantly impacts the yield and functionality of recombinant UQCRFS1:

How can researchers verify the proper incorporation of the 2Fe-2S cluster in recombinant UQCRFS1?

Verification of proper 2Fe-2S cluster incorporation requires several complementary approaches:

• UV-Visible Spectroscopy: Properly assembled Fe-S clusters display characteristic absorption peaks; the 2Fe-2S cluster in UQCRFS1 shows distinctive absorbance features between 320-500 nm.

• EPR Spectroscopy: Electron paramagnetic resonance provides detailed information about the electronic structure and environment of the Fe-S cluster, confirming proper coordination.

• Mössbauer Spectroscopy: This technique provides information about the oxidation state and chemical environment of iron atoms in the cluster.

• Circular Dichroism: Near-UV and visible CD spectra can confirm proper cluster incorporation and protein folding.

• Activity Assays: Functional assays measuring electron transfer capability indicate whether the incorporated cluster is catalytically active.

• Metal Quantification: ICP-MS (Inductively Coupled Plasma Mass Spectrometry) can quantify iron content, with a 2:1 iron-to-protein ratio expected for properly reconstituted UQCRFS1.

• Protein Stability: Thermal shift assays comparing apo-protein to holo-protein can confirm cluster-mediated stabilization.

The BCS1L-mediated incorporation of UQCRFS1 with its 2Fe-2S cluster into the pre-CIII2 complex is essential for rendering it catalytically active . Therefore, verification of proper cluster incorporation is critical for ensuring the functionality of recombinant UQCRFS1 in experimental systems.

What are the most promising research applications for recombinant Lagothrix lagotricha UQCRFS1?

Recombinant Lagothrix lagotricha UQCRFS1 offers several promising research applications:

• Comparative Mitochondrial Biology: As a non-human primate model, it provides an evolutionary perspective on respiratory complex function and assembly between human and other mammalian systems.

• Structural Biology: The protein can be used for crystallographic or cryo-EM studies to elucidate species-specific aspects of Complex III architecture.

• Disease Modeling: It serves as a comparative model for studying mechanisms of UQCRFS1-related mitochondrial disorders, which present with symptoms including lactic acidosis, cardiomyopathy, and alopecia totalis .

• Therapeutic Development: The protein can be used to screen compounds that might stabilize mutant UQCRFS1 or enhance residual Complex III activity.

• Evolutionary Medicine: Studying differences between human and woolly monkey UQCRFS1 might reveal adaptive changes in mitochondrial function relevant to human disease.

The recent identification of bi-allelic UQCRFS1 variants associated with mitochondrial disorders highlights the clinical relevance of this research, while the complex phylogeography of Lagothrix lagotricha provides an evolutionary context that enriches comparative studies.

What methodological advances are needed to better study UQCRFS1 and Complex III disorders?

Several methodological advances would enhance UQCRFS1 research:

• Improved Fe-S Cluster Reconstitution Protocols: More efficient and consistent methods for in vitro and in vivo Fe-S cluster assembly would advance structural and functional studies.

• Tissue-Specific Models: Development of organ-specific models (especially cardiac and neuronal) to study tissue-specific effects of UQCRFS1 deficiency.

• High-Resolution Structural Studies: Advanced cryo-EM techniques to visualize the dynamic process of UQCRFS1 incorporation into Complex III.

• In Situ Processing Assays: Methods to study the unusual in situ processing of UQCRFS1 after its incorporation into Complex III .

• Single-Cell Energetics: Techniques to assess mitochondrial function in individual cells with UQCRFS1 mutations.

• Pharmacological Chaperones: Development of compounds that could stabilize mutant UQCRFS1 proteins.

• Gene Therapy Approaches: Methods for delivering functional UQCRFS1 to affected tissues.

The unique aspect of UQCRFS1 biology—where its cleaved presequence remains as a functional component of Complex III rather than being degraded —represents a particularly intriguing area for methodological innovation. Understanding this dual functionality could provide insights into both protein trafficking and respiratory complex assembly.