Recombinant Candida glabrata Ribosome-recycling factor, mitochondrial (RRF1), partial

What is the function of Ribosome-recycling factor (RRF1) in Candida glabrata?

RRF1 encodes the mitochondrial ribosome recycling factor (Rrf1p) that is essential for mitochondrial protein synthesis and maintaining respiratory function. The protein is encoded by the nuclear genome but functions within mitochondria. Experimental evidence from deletion studies demonstrates that without RRF1, C. glabrata cells become respiratory incompetent and cannot grow on non-fermentable carbon sources. Rrf1p facilitates the disassembly of ribosomes at the termination of protein synthesis, allowing ribosomal components to be recycled for new rounds of translation .

How is RRF1 involved in mitochondrial genome stability?

RRF1 is critical for maintaining mitochondrial genome integrity. Research using RRF1 deletion strains shows that loss of this gene leads to irreversible damage to the mitochondrial genome, resulting in respiratory incompetence and conversion to cytoplasmic petites (rho-/rho0 mutants). When visualized with DAPI staining, mitochondrial DNA in Δrrf1 cells appears diffuse and less abundant compared to wild-type controls . Importantly, once mitochondrial DNA damage has occurred, simply reintroducing RRF1 on a plasmid cannot restore respiratory function, indicating that RRF1 plays a preventative rather than curative role in maintaining mitochondrial genome stability .

What experimental approaches are commonly used to study RRF1 function?

Several complementary approaches are employed to study RRF1 function:

• Gene deletion studies where the complete RRF1 ORF is replaced with marker genes (e.g., TRP1)

• Plasmid-based complementation using low or high copy number vectors carrying RRF1

• Growth phenotype analysis on fermentable (glucose) versus non-fermentable (glycerol, ethanol) carbon sources

• Protein localization via western blotting with specific antibodies

• Temperature-sensitive mutations (e.g., rrf1-L209P) that allow conditional inactivation

• Analysis of mitochondrial protein synthesis rates at different temperatures

These approaches have revealed that Rrf1p localizes to mitochondria and is processed to a smaller size (23 kDa) than predicted from its sequence (26.4 kDa), suggesting removal of a mitochondrial targeting sequence .

How do temperature-sensitive mutations in RRF1 affect mitochondrial function?

This selective inhibition allows researchers to temporally control RRF1 function and study immediate consequences of its loss without the confounding effects of complete mitochondrial genome destabilization that occurs in deletion strains. The rrf1-L209P mutation is particularly useful because it provides a clean separation of phenotypes - normal growth at 30°C with complete respiratory deficiency at 36°C .

What are the challenges in studying RRF1 deletion phenotypes?

Studying RRF1 deletion presents significant challenges due to the irreversible nature of mitochondrial damage upon gene deletion. Key experimental obstacles include:

• Irreversible mitochondrial genome damage - Once RRF1 is deleted, mitochondrial DNA becomes unstable and is progressively lost, creating rho-/rho0 petites

• Secondary effects masking primary function - The cascading effects of mitochondrial dysfunction make it difficult to distinguish direct from indirect consequences of RRF1 deletion

• Inability to rescue with plasmid-borne RRF1 after deletion - Simple reintroduction of the gene cannot restore function once mitochondrial DNA is damaged

To overcome these challenges, researchers have developed specialized approaches:

• Creating heterozygous diploids (RRF1/Δrrf1) and obtaining haploid deletion strains through sporulation while maintaining a plasmid-borne copy of RRF1

• Using temperature-sensitive alleles like rrf1-L209P that allow controlled inactivation

• Employing regulatable promoters to allow titratable expression of RRF1

These approaches have revealed that RRF1 function is essential for mitochondrial protein synthesis, and without it, irreversible damage to the mitochondrial genome occurs .

How does the structure of Rrf1p relate to its function in mitochondrial ribosomes?

The temperature-sensitive phenotype of the rrf1-L209P mutation provides insights into structure-function relationships. The L209P substitution likely disrupts the protein's secondary structure since proline introduces kinks in alpha-helices. This structural disruption becomes critical at higher temperatures (36°C) while permitting sufficient function at lower temperatures (30°C) .

Comparative analysis with other ribosome recycling factors suggests that Rrf1p likely adopts a two-domain structure similar to bacterial RRF. The first domain mimics tRNA structure to interact with the ribosomal A-site, while the second domain interfaces with ribosomal components to facilitate recycling. The L209P mutation may disrupt interdomain interactions or interfere with binding to mitochondrial ribosomes at higher temperatures .

What is the relationship between RRF1 function and antifungal resistance in C. glabrata?

While direct evidence linking RRF1 to antifungal resistance is limited, several connections can be made:

• Mitochondrial function influences stress responses, which in turn affect antifungal susceptibility

• C. glabrata displays both innate and acquired resistance to azole antifungals, with mitochondrial dysfunction proposed as an adaptive strategy for survival

• Pdr1, a transcription factor regulating drug resistance in C. glabrata, is activated by mitochondrial dysfunction

Disruptions in mitochondrial protein synthesis through RRF1 inactivation could potentially alter cellular responses to antifungals. In particular, recent research indicates that transient mitochondrial dysfunction may be an adaptive strategy for C. glabrata survival in the presence of fluconazole and within macrophage phagosomes .

How can researchers effectively express and purify recombinant C. glabrata RRF1?

When producing recombinant C. glabrata RRF1 for in vitro studies, several factors must be considered:

• Expression system selection - Bacterial systems like E. coli offer high yield but may lack appropriate post-translational modifications, while yeast-based systems might provide more authentic processing

• Signal sequence management - The N-terminal mitochondrial targeting sequence (approximately 3.4 kDa based on observed vs. predicted size) should be omitted from recombinant constructs to prevent aggregation and improper folding

• Tag selection - Affinity tags facilitate purification but may affect activity, requiring comparison with untagged versions

• Solubility optimization - Co-expression with chaperones or use of solubility-enhancing fusion partners

• Purification strategy - Multi-step purification including affinity chromatography followed by ion exchange and size exclusion

Western blot analysis has shown that mature Rrf1p has an apparent molecular mass of 23 kDa, smaller than the 26.4 kDa predicted from the gene sequence, indicating processing of the mitochondrial targeting sequence . This information is crucial when designing expression constructs for the mature, functional protein.

How can researchers study RRF1 function without triggering irreversible mitochondrial damage?

To study RRF1 function while preserving mitochondrial integrity, researchers should implement:

• Temperature-sensitive alleles like rrf1-L209P that enable rapid and reversible inactivation

• Creation of heterozygous diploids followed by tetrad dissection with plasmid-borne wild-type RRF1

• Time-course experiments that capture early events following RRF1 inactivation before irreversible damage

• Comparative analyses with other mitochondrial translation factors to establish specific roles

The experimental approach used by Teyssier et al. demonstrates the value of these strategies - they generated a haploid Δrrf1 strain maintained by plasmid-borne RRF1 from a heterozygous diploid using sporulation techniques. This approach successfully maintained intact mitochondria despite genomic deletion of RRF1 .

What assays can determine the specific impact of RRF1 on mitochondrial translation?

To assess RRF1's specific role in mitochondrial translation, researchers can employ:

• In vivo labeling of mitochondrial translation products using 35S-methionine in the presence of cycloheximide (to inhibit cytoplasmic translation)

• Measurement of oxygen consumption rates as a proxy for respiratory chain function

• Analysis of mitochondrial ribosome profiles to detect accumulation of post-termination complexes

• Comparison of mitochondrial versus cytoplasmic translation rates at different temperatures in temperature-sensitive mutants

These approaches have demonstrated that in the rrf1-L209P mutant at 36°C, mitochondrial protein synthesis is inhibited by 90% while cytoplasmic protein synthesis remains unaffected, confirming RRF1's specific role in mitochondrial translation .

How does RRF1 in C. glabrata compare to homologs in other fungi?

Ribosome recycling factors are conserved across species, from bacteria to eukaryotes, though with specific adaptations:

• The nuclear-encoded RRF1 gene in C. glabrata shows significant sequence similarity to Escherichia coli ribosome recycling factor

• Similar to Saccharomyces cerevisiae, C. glabrata RRF1 contains an N-terminal mitochondrial targeting sequence that is cleaved upon import

• The mature Rrf1p functions specifically in mitochondrial ribosomes, which more closely resemble bacterial ribosomes than cytoplasmic eukaryotic ribosomes

This conservation reflects the endosymbiotic origin of mitochondria from bacteria and explains why mitochondrial translation machinery components often resemble bacterial counterparts more than their cytoplasmic eukaryotic equivalents .

What does RRF1 research reveal about mitochondrial evolution in pathogenic yeasts?

Research on RRF1 provides insights into mitochondrial evolution in pathogenic yeasts:

• The essential nature of RRF1 for mitochondrial function even in a facultative anaerobe like C. glabrata highlights the continued importance of mitochondria beyond ATP production

• The ability of C. glabrata to survive RRF1 deletion (albeit with respiratory deficiency) contrasts with the lethality of similar deletions in many other organisms

• The conversion to petite (rho-/rho0) phenotypes upon RRF1 loss represents an extreme but viable adaptation

These findings suggest that while mitochondrial function remains important in C. glabrata, the organism has evolved mechanisms to survive significant mitochondrial dysfunction, which may contribute to its adaptability as a pathogen .

How does mitochondrial function impact C. glabrata virulence and pathogenesis?

Mitochondrial function, including processes dependent on RRF1, may influence C. glabrata virulence in several ways:

• Adaptability to host environments - The ability to modulate mitochondrial function may help C. glabrata survive in diverse host niches with varying nutrient availability

• Stress responses - Mitochondrial signaling affects cellular stress responses, which are critical during host infection

• Metabolic flexibility - The capacity to shift between fermentative and respiratory metabolism contributes to survival in changing host environments

Recent research suggests that transient mitochondrial dysfunction may be an adaptive strategy for C. glabrata survival within phagosomes of macrophages . Since RRF1 is essential for mitochondrial translation, it plays an indirect but potentially important role in pathogenesis by maintaining mitochondrial function.

Could RRF1 serve as a potential antifungal target?

Several factors make RRF1 an interesting potential target for antifungal development:

• Essential function - RRF1 is required for respiratory competence in C. glabrata

• Structural distinctiveness - As a mitochondrial protein with bacterial origins, Rrf1p likely differs structurally from human cytoplasmic translation factors

• Specificity opportunity - Differences between fungal and human mitochondrial translation systems might allow selective targeting

• C. glabrata can survive as a respiratory-deficient petite mutant

• The high degree of conservation between fungal and human mitochondrial translation machinery

• Potential off-target effects on human mitochondrial translation

The temperature-sensitive rrf1-L209P mutation provides proof-of-principle that targeted disruption of RRF1 function is possible , though developing clinically viable inhibitors would require careful optimization to achieve fungal selectivity.