Recombinant Candida glabrata 26S proteasome regulatory subunit RPN11 (RPN11)

What is the functional significance of RPN11 in Candida glabrata?

RPN11 in Candida glabrata likely serves multiple essential functions similar to its homologs in other yeast species. As a 26S proteasome regulatory subunit, it primarily functions as a deubiquitinating enzyme within the lid of the proteasome complex, essential for protein degradation pathways. Research in related yeast species has shown that the N-terminal region contains the catalytic deubiquitinating activity through the MPN+/JAMM motif, while the C-terminal domain plays crucial roles in maintaining mitochondrial morphology, function, and proper cell cycle regulation . The dual functionality of RPN11 suggests its importance in both proteasomal degradation pathways and mitochondrial dynamics in C. glabrata, potentially contributing to its pathogenicity and stress response mechanisms.

How can I generate deletion mutants of RPN11 in Candida glabrata for functional studies?

To generate RPN11 deletion mutants in C. glabrata, researchers can employ a PCR-based gene disruption strategy using selectable markers. Based on established protocols, you can:

• Design primers containing 40-50bp homology to the regions flanking the RPN11 gene and a sequence for amplifying a selectable marker (e.g., hygromycin resistance cassette).

• PCR amplify the disruption cassette.

• Transform C. glabrata (e.g., BG2 strain) with the purified PCR product.

• Select transformants on appropriate antibiotic-containing media (e.g., hygromycin).

• Confirm deletion by PCR using primers specific to the RPN11 internal regions.

For example, primers similar to those used in relevant studies (5'-TGGTGCTTTGGACGCTACAT-3' & 5'-TCATCGCAAAAGCAATTGGACA-3') can be employed to verify deletion . For complementation studies, the RPN11 ORF can be cloned into an appropriate expression vector, such as a CEN/ARS episomal plasmid, under a constitutive promoter.

What methods are available to study the subcellular localization of RPN11 in Candida glabrata?

Several complementary approaches can be used to determine RPN11 subcellular localization:

• Fluorescent protein tagging: Generate a C. glabrata strain expressing RPN11 fused to GFP or another fluorescent protein. This enables visualization of the protein's location using fluorescence microscopy under different growth conditions.

• Subcellular fractionation: Separate cellular components through differential centrifugation and density gradient techniques. This approach has successfully demonstrated that a small portion of Rpn11 associates with mitochondria in S. cerevisiae, with this association being more pronounced in the rpn11-m1 mutant .

• Immunofluorescence microscopy: Use specific antibodies against RPN11 or epitope-tagged versions (e.g., 3XHA-tagged RPN11) for immunolocalization studies.

• Biochemical co-purification: Isolate specific organelles (proteasomes, mitochondria) and analyze the presence of RPN11 by Western blotting.

These methods can collectively provide a comprehensive understanding of RPN11's dynamic localization during different growth phases and stress conditions.

How does RPN11 expression in Candida glabrata change during host-pathogen interaction?

While specific data on RPN11 expression during host-pathogen interaction in C. glabrata is limited in the provided search results, researchers can employ RNA polymerase II (RNAPII) ChIP-seq to analyze transcriptional responses during macrophage infection. This approach has been successfully used to map genome-wide transcriptional changes in C. glabrata during macrophage infection with high temporal resolution .

To study RPN11 expression specifically:

• Perform RNAPII ChIP-seq at different time points during macrophage infection.

• Analyze the RNAPII occupancy at the RPN11 locus to determine its transcriptional activity.

• Compare RPN11 expression patterns with known temporal transcriptional responses.

• Validate findings using RT-qPCR or reporter gene assays.

This approach would reveal whether RPN11 shows dynamic expression during the chronological response to macrophage infection, similar to the patterns observed for other genes involved in virulence and stress response.

What is the role of the C-terminal domain of RPN11 in mitochondrial function in Candida glabrata?

Based on studies in S. cerevisiae, the C-terminal domain of RPN11 is critical for maintaining normal mitochondrial morphology and function. In the rpn11-m1 mutant lacking the last 31 amino acids, significant mitochondrial defects are observed, while mutations in the catalytic MPN+/JAMM motif do not affect mitochondrial phenotypes .

To investigate this in C. glabrata, researchers should:

• Generate targeted mutations in the C-terminal domain of C. glabrata RPN11, creating truncations similar to rpn11-m1.

• Assess mitochondrial morphology using fluorescent markers (e.g., MitoTracker dyes or mitochondria-targeted GFP).

• Measure mitochondrial function through respiratory capacity assays, ROS production, and membrane potential analysis.

• Perform complementation experiments by expressing the C-terminal domain in trans.

In S. cerevisiae, expression of the last 100 amino acids of Rpn11 can suppress the mitochondrial defects of rpn11-m1 mutants . Determining if this holds true for C. glabrata would establish whether the mechanism of RPN11's involvement in mitochondrial dynamics is conserved across yeast species and may reveal pathogen-specific adaptations.

How does RPN11 contribute to antifungal resistance in Candida glabrata?

While direct evidence linking RPN11 to antifungal resistance in C. glabrata is not explicitly stated in the provided search results, we can design experiments to investigate this relationship based on related findings:

• Generate RPN11 mutants (particularly C-terminal domain mutants) and test their susceptibility to various antifungals, especially azoles, using standardized minimum inhibitory concentration (MIC) assays.

• Perform transcriptomic analysis of wild-type and RPN11 mutant strains under antifungal stress to identify differentially expressed genes related to drug resistance.

• Investigate potential regulatory interactions between RPN11 and known resistance factors such as CgXbp1, which has been shown to affect fluconazole resistance and regulate genes associated with drug resistance .

• Assess proteasome assembly and function in RPN11 mutants, as altered proteasomal degradation could affect the levels of proteins involved in drug efflux or detoxification.

A comprehensive analysis combining these approaches would establish whether RPN11 directly contributes to the intrinsically high azole resistance observed in C. glabrata.

What is the relationship between RPN11 and proteasome storage granule (PSG) formation in Candida glabrata during stationary phase?

In S. cerevisiae, the integrity of Rpn11 is critical for proteasome storage granule (PSG) formation during stationary phase. The rpn11-m1 mutant is unable to form PSGs and shows reduced viability in stationary phase . To investigate whether a similar mechanism exists in C. glabrata:

• Generate fluorescently tagged proteasome subunits (e.g., Rpn5-GFP) in wild-type and RPN11 mutant C. glabrata strains.

• Monitor proteasome localization during exponential and stationary phases using fluorescence microscopy.

• Assess whether PSG formation occurs in C. glabrata and if RPN11 mutations affect this process.

• Determine the correlation between PSG formation, proteasome activity, and long-term survival in C. glabrata.

• Investigate whether the C-terminal domain of C. glabrata RPN11 can complement PSG formation defects when expressed in trans.

This table summarizes findings from S. cerevisiae that could guide similar investigations in C. glabrata to determine conservation of RPN11 function in PSG formation.

How do RPN11 mutations affect Candida glabrata virulence in macrophage infection models?

To assess the impact of RPN11 mutations on C. glabrata virulence during macrophage interactions:

• Generate C. glabrata strains with various RPN11 mutations, particularly focusing on the C-terminal domain.

• Perform macrophage infection assays using different time points (0.5h to 24h) to capture the dynamic host-pathogen interaction.

• Measure fungal survival rates within macrophages by colony-forming unit (CFU) assays.

• Employ RNAPII ChIP-seq to analyze transcriptional profiles of wild-type and RPN11 mutant strains during macrophage infection, similar to studies with CgXbp1 .

• Assess key virulence phenotypes such as phagocytosis resistance, phagosome maturation inhibition, and macrophage escape.

• Validate findings using in vivo infection models such as Galleria mellonella, which has been successfully used to study C. glabrata virulence .

These approaches would reveal whether RPN11, particularly its C-terminal domain, plays important roles in C. glabrata pathogenicity similar to transcription factors like CgXbp1.

What are the protein-protein interaction networks of RPN11 in Candida glabrata and how do they differ from other fungal species?

Understanding RPN11's interactome would provide insights into its functions beyond the proteasome. To map and compare protein-protein interactions:

• Perform immunoprecipitation coupled with mass spectrometry (IP-MS) using epitope-tagged RPN11 in C. glabrata.

• Use proximity-dependent biotin identification (BioID) or related techniques to identify proteins in the vicinity of RPN11, particularly at the mitochondria.

• Compare interaction networks between wild-type RPN11 and C-terminal mutants to identify domain-specific interactions.

• Conduct cross-species comparative analyses between C. glabrata, S. cerevisiae, and C. albicans to identify conserved and species-specific interactions.

Evidence from S. cerevisiae suggests that the C-terminal domain of Rpn11 may function independently of the proteasome and does not interact directly with other proteasome subunits like Rpn8 . Determining whether similar interaction patterns exist in C. glabrata would provide mechanistic insights into RPN11's role in various cellular processes, particularly those related to pathogenicity and drug resistance.