Recombinant Saccharomyces cerevisiae Cytochrome b-c1 complex subunit 9 (QCR9)

What is QCR9 and what is its structural composition?

QCR9 is a nuclear gene in Saccharomyces cerevisiae that encodes the 7.3-kDa subunit 9 of the mitochondrial cytochrome bc1 complex. The gene includes a 195-base pair open reading frame capable of encoding a protein of 66 amino acids with a predicted molecular weight of 7471 Da . Notably, the N-terminal methionine of subunit 9 is removed posttranslationally, as the N-terminal sequence of the purified protein begins with serine at position 2 .

The genomic structure of QCR9 includes a 213-base pair intron that separates the ATG triplet corresponding to the N-terminal methionine from the rest of the open reading frame. This intron contains the standard 5' donor, 3' acceptor, and TACTAAC sequences necessary for splicing in yeast . Interestingly, the QCR9 intron contains a nucleotide sequence with high similarity (15 out of 18 nucleotides) to a sequence in the intron of COX4, suggesting possible evolutionary relationships between these mitochondrial protein genes .

How is QCR9 evolutionarily conserved across species?

Analysis of QCR9 reveals significant evolutionary conservation. The deduced amino acid sequence of yeast subunit 9 shares 39% identity with its homolog from beef heart cytochrome bc1 complex . When considering conservative amino acid substitutions, the similarity increases to 56%, indicating functional conservation across diverse eukaryotes .

Secondary structure prediction of the 7.3-kDa protein reveals a single possible transmembrane helix. In this helix, the amino acids conserved between beef heart and yeast are asymmetrically arranged along one face, suggesting this domain is involved in a conserved interaction with another hydrophobic protein within the cytochrome bc1 complex . This conservation pattern points to a critical functional role that has been maintained through evolutionary divergence.

What experimental techniques are most effective for QCR9 isolation?

For effective isolation of QCR9, researchers have successfully employed several complementary techniques:

• Genomic library screening: QCR9 was initially isolated from a yeast genomic library using hybridization with a degenerate oligonucleotide corresponding to nine amino acids proximal to the N-terminus of purified subunit 9 .

• cDNA isolation: To confirm splice junctions and the 5' end of the message, researchers isolated and sequenced a cDNA copy of QCR9 .

• Protein purification: For studying the protein directly, isolation of the mitochondrial cytochrome bc1 complex followed by subunit separation has been effective in obtaining purified QCR9 protein.

For expression analysis, researchers have successfully used real-time PCR with gene-specific primers, with QCR9 sometimes serving as a reference gene for normalization . When designing primers, researchers should account for the intronic structure of the gene to avoid interference with expression analysis.

What phenotypes result from QCR9 deletion in S. cerevisiae?

Deletion of QCR9 in S. cerevisiae produces several distinct and measurable phenotypes:

• Growth deficiency: Strains with QCR9 deletion (such as JDP1 and JDP2) grow very poorly or not at all on non-fermentable carbon sources, demonstrating the critical role of QCR9 in respiratory growth .

• Enzymatic activity loss: QCR9 deletion strains exhibit at most only 5% of wild-type ubiquinol-cytochrome c oxidoreductase activity, indicating that this subunit is essential for proper enzyme function .

• Spectral alterations: Optical spectra of mitochondrial membranes from QCR9 deletion strains show diminished cytochrome b absorption, similar to the spectra of membranes from strains lacking the Rieske iron-sulfur protein . This suggests an interaction between subunit 9, the iron-sulfur protein, and cytochrome b.

• EPR signal loss: EPR spectroscopy of membranes from QCR9 deletion strains indicates that the g = 1.90 signal characteristic of the Rieske iron-sulfur cluster is absent, even though mature-sized apoprotein is present .

• Cytochrome c1 reduction kinetics: Pre-steady state reduction of cytochrome c1 is markedly slowed, but not eliminated, in QCR9 deletion strains .

Importantly, complementation studies have shown that when JDP1 and JDP2 deletion strains are transformed with a plasmid carrying QCR9, the resulting yeast grow normally on ethanol/glycerol and exhibit normal cytochrome c reductase activities and optical spectra, confirming the specific role of QCR9 in the observed phenotypes .

How does QCR9 interact with other subunits of the cytochrome bc1 complex?

QCR9 plays a crucial role in the assembly and stabilization of the cytochrome bc1 complex through multiple protein-protein interactions:

• Core structure interactions: QCR9 appears to be assembled around a core comprised of cytochrome b, subunit 7, and subunit 8. When any of these core components is deleted, the others are also lost, indicating their interdependence .

• Iron-sulfur protein interaction: Evidence suggests a direct interaction between QCR9 and the Rieske iron-sulfur protein. In the absence of subunit 9, the conformation of the iron-sulfur protein is altered such that the protein becomes more labile, the iron-sulfur cluster is not properly inserted, and its interaction with cytochrome b is modified in a manner that distorts the heme environment .

• Cytochrome b interaction: The diminished cytochrome b absorption in QCR9 deletion strains is not due to impaired synthesis of cytochrome b, but rather to a post-assembly effect on the heme environment resulting from the absence of subunit 9 .

• Subcomplex formation: Research on deletion mutants suggests potential subcomplexes between subunit 6, subunit 9, and cytochrome c1, indicating that QCR9 may participate in multiple interaction networks within the larger complex .

These interactions highlight QCR9's critical role in both the assembly and stability of the cytochrome bc1 complex, despite its small size.

What methods are recommended for studying QCR9 expression?

For accurate analysis of QCR9 expression, several validated methodological approaches can be employed:

Real-Time PCR Protocol:

• Extract total RNA from yeast cultures using standard protocols

• Perform reverse transcription using random hexamer oligonucleotides

• Conduct real-time PCR in 384-well plates with the following reaction mixture:

  • 5 μl of 2x SYBR Fast qPCR MasterMix

  • 3 μl of 1 mM forward and reverse gene-specific primers mix

  • 2 μl of cDNA

The amplification protocol should include:

• Preincubation at 95°C for 3 min

• 45 cycles at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s

• Melting curve analysis from 65°C to 97°C at 2.2°C/s

For normalization, researchers have successfully used reference genes such as U2 spliceosomal RNA and small cytosolic RNA (SCR1), with QCR9 itself sometimes serving as a reference gene due to its stable expression in certain conditions . The NormFinder algorithm can be applied to identify the best reference genes for specific experimental conditions.

For protein-level analysis, Western blotting of HA-tagged constructs has proven effective, with samples prepared by either rapid freezing in liquid nitrogen or quenching with trichloroacetic acid solution to preserve protein integrity .

How can CRISPR-Cas9 be utilized for precise engineering of QCR9 in S. cerevisiae?

CRISPR-Cas9 offers powerful tools for precise genetic manipulation of QCR9 in S. cerevisiae. Implementation requires several key components and considerations:

Components for QCR9 Editing:

• Cas9 expression: Typically delivered through a plasmid with appropriate promoter (often constitutive)

• gRNA design and expression: For QCR9 targeting, design a 20-nucleotide guide sequence upstream of a PAM site (NGG) within the gene. Expression via RNA polymerase III promoters, particularly the SNR52 promoter with SUP4 terminator, has shown high efficiency in yeast

• Donor DNA template: Design repair templates with homology arms flanking the desired modification site

Experimental Protocol:

• gRNA design: Identify target sites within QCR9 using computational tools to minimize off-target effects

• Template design: Create donor DNA with desired modifications (point mutations, insertions, deletions) and 40-60 bp homology arms, ensuring PAM site modification to prevent continuous cutting

• Co-transformation: Transform yeast with Cas9-expressing plasmid, gRNA-expressing plasmid, and repair template

• Screening: Verify successful editing through PCR, sequencing, or functional assays

For QCR9 knockout studies, efficiencies approaching 100% have been achieved in laboratory strains . For more complex edits, including precise mutations or insertions, efficiencies may be lower but still practical for research purposes. The system can be combined with in vivo assembly of various DNA fragments, eliminating the need for separate cloning processes .

How does QCR9 deletion affect mitochondrial function and iron-sulfur protein assembly?

QCR9 deletion has profound effects on mitochondrial function through several interconnected mechanisms:

• Iron-sulfur protein conformation: In QCR9 deletion strains, the Rieske iron-sulfur protein is present in normal amounts and processed to its mature form, but shows increased lability to endogenous proteases during membrane isolation . This suggests that QCR9 plays a role in maintaining the proper conformation and stability of the iron-sulfur protein.

• Iron-sulfur cluster formation: EPR spectroscopy reveals the absence of the characteristic g = 1.90 signal of the Rieske iron-sulfur cluster in QCR9 deletion strains . This indicates that without QCR9, the iron-sulfur cluster is either not properly inserted or exists in an altered, EPR-silent state.

• Electron transfer kinetics: Pre-steady state reduction of cytochrome c1 is significantly slowed in QCR9 deletion strains . This suggests the presence of a sluggishly reactive derivative of the iron-sulfur cluster that can still participate in electron transfer but at greatly reduced rates.

• Cytochrome b environment: The absence of QCR9 alters the heme environment of cytochrome b, as evidenced by diminished absorption spectra . This occurs despite normal synthesis of cytochrome b, indicating that QCR9 plays a post-assembly role in maintaining the proper conformation of the complex.

These findings collectively suggest that QCR9, despite being a small "supernumerary" subunit, plays a critical role in maintaining the structural integrity and functional capability of the cytochrome bc1 complex, particularly in relation to the iron-sulfur protein and its redox-active cluster.

What is the role of QCR9 in the assembly pathway of the cytochrome bc1 complex?

The assembly of the cytochrome bc1 complex in S. cerevisiae follows a specific pathway in which QCR9 plays a defined role:

• Core assembly: The initial assembly involves cytochrome b, subunit 7, and subunit 8, which form the core around which other subunits are assembled . Deletion of any one of these three components causes the loss of the other two, indicating their interdependence.

• QCR9 incorporation: QCR9 is assembled around this core structure, where it appears to play a role in stabilizing or facilitating the incorporation of other components.

• Subcomplex formation: Evidence of interactions between subunit 6, subunit 9 (QCR9), and cytochrome c1 suggests that these components may form a subcomplex during the assembly process .

• Independent assembly of core proteins: Core protein 1 and core protein 2 show coordinated presence in mitochondrial membranes, suggesting they can form a subcomplex independent of other subunits .

• Incomplete complex in QCR9 absence: In the absence of QCR9, cytochrome c1, iron-sulfur protein, core protein 1, core protein 2, and subunit 9 can still be assembled in the membrane, albeit in reduced amounts, while subunit 6 is lost .

This assembly pathway indicates that QCR9, while not part of the core structure, plays a critical role in the proper assembly and stability of the complete functional complex. The ability of certain subcomplexes to form in the absence of QCR9 suggests a modular assembly process with QCR9 serving as a key connector or stabilizer between different modules.

Can QCR9 be utilized as a reference gene in S. cerevisiae gene expression studies?

QCR9 has been evaluated as a potential reference gene for normalization in gene expression studies in S. cerevisiae:

Validation Protocol:

• Extract RNA from all experimental conditions

• Perform real-time PCR for candidate reference genes including QCR9

• Analyze stability using NormFinder or similar algorithm

• Calculate normalization factors using the Vandesompele method if multiple reference genes are selected

When considering QCR9 as a reference gene, researchers should be aware that its expression might be affected in experiments involving mitochondrial function, respiratory growth, or energy metabolism, given its role in the respiratory chain.

How can QCR9 research contribute to understanding mitochondrial diseases?

Research on QCR9 in S. cerevisiae provides valuable insights for understanding mitochondrial diseases through several avenues:

• Model system: S. cerevisiae serves as an excellent model for studying conserved aspects of mitochondrial function and assembly. The QCR9 gene, with its 56% similarity to mammalian counterparts when considering conservative substitutions, offers insights into conserved mechanisms of cytochrome bc1 complex assembly and function .

• Disease mechanisms: Cytochrome bc1 complex deficiencies in humans lead to mitochondrial diseases with varied phenotypes. Studying how QCR9 deletion affects complex assembly, stability, and function in yeast provides models for understanding pathogenic mechanisms in human diseases caused by mutations in complex III components.

• Protein-protein interactions: The research showing QCR9's role in iron-sulfur protein stability and proper incorporation of the iron-sulfur cluster offers insights into potential therapeutic approaches targeting protein-protein interactions in mitochondrial disease .

Future research directions could include creating humanized yeast strains expressing mammalian homologs of QCR9 to directly test functional conservation and potential disease-causing mutations.

What are the potential applications of QCR9 in recombinant S. cerevisiae systems?

QCR9 knowledge can be leveraged in various applications involving recombinant S. cerevisiae:

• Biofuel production: Engineering the cytochrome bc1 complex through QCR9 modification could potentially enhance respiratory capacity and energy efficiency in yeast strains used for biofuel production.

• Metabolic engineering: Understanding the role of QCR9 in respiratory function provides targets for manipulating energy metabolism in engineered yeast strains for improved production of various compounds.

• Protein expression systems: Recombinant S. cerevisiae expressing modified versions of QCR9 could be used as platforms for studying complex III assembly and function, particularly for testing the effects of mutations found in human disease.

• Vaccine development: Whole, heat-killed, recombinant S. cerevisiae has been used as a vehicle for vaccine development . Understanding mitochondrial function through QCR9 research could potentially contribute to optimizing these systems.

As CRISPR-Cas9 technology continues to advance, precise engineering of QCR9 and other components of the cytochrome bc1 complex will become increasingly feasible, opening new avenues for applied research in biotechnology and medicine.