Recombinant Candida glabrata ATP-dependent RNA helicase FAL1 (FAL1)

What is FAL1 and what is its significance in Candida glabrata?

FAL1 (ATP-dependent RNA helicase FAL1) is a crucial DEAD-box RNA helicase in Candida glabrata that plays an essential role in ribosome biogenesis, particularly in the assembly of the small subunit (SSU). As a member of the DEAD-box protein family, it contains the characteristic Asp-Glu-Ala-Asp (DEAD) motif and functions in RNA metabolism through ATP-dependent RNA unwinding activities. FAL1 is particularly significant because it is involved in the early maturation steps of 18S rRNA, which is necessary for proper ribosome function in this clinically important fungal pathogen .

What are the optimal storage conditions for recombinant FAL1 protein?

Recombinant FAL1 protein stability depends on several factors including buffer composition, storage temperature, and formulation. For optimal shelf life and activity:

For reconstitution, it is recommended to briefly centrifuge the vial prior to opening, and after reconstitution, the protein should be aliquoted to minimize freeze-thaw cycles .

How does FAL1 compare structurally to other DEAD-box RNA helicases?

FAL1 shares the conserved structural organization of DEAD-box helicases, consisting of:

• Two RecA-like domains connected by a flexible hinge region

• Conserved motifs for ATP binding and hydrolysis, including the signature DEAD sequence

• RNA-binding regions primarily in the C-terminal domain

The full-length protein consists of 399 amino acids with specific sequence features that contribute to its function in ribosome assembly. Unlike some helicases that may contain additional domains for specific functions, FAL1 maintains a relatively conserved helicase core structure, which is likely optimized for its specialized role in SSU biogenesis .

How should I design experiments to characterize the ATPase activity of FAL1?

When designing experiments to measure FAL1's ATPase activity, consider the following experimental protocol based on published research:

Materials and Methods:

• Purified recombinant His-tagged FAL1 protein (wild-type and mutant variants)

• ATP substrate and RNA (for stimulation of activity)

• Controls: No-protein control and catalytically inactive mutant (e.g., DQAD mutation)

Experimental Design:

• Include conditions both with and without RNA to demonstrate RNA-dependent stimulation

• Use a concentration series of ATP to determine kinetic parameters

• Include time course measurements to establish linear reaction rates

Key Measurements:

• ATP hydrolysis rate (with and without RNA)

• Effect of RNA concentration on ATPase activity

• Impact of mutations on catalytic activity

Research has shown that wild-type FAL1 exhibits minimal ATPase activity in the absence of RNA, but this activity is stimulated >3-fold in the presence of RNA. The DQAD mutation can serve as a negative control as it almost completely abolishes catalytic activity .

What experimental approaches can demonstrate the interaction between FAL1 and Sgd1?

Based on successful published research, the following experimental designs are recommended to study the FAL1-Sgd1 interaction:

Co-Immunoprecipitation from Cell Extracts:

• Express tagged versions of FAL1 (e.g., HA-tagged) in yeast cells

• Immobilize MBP-Sgd1-His on amylose resin

• Incubate with yeast cell extracts (with and without RNase treatment)

• Analyze co-precipitated proteins by Western blot

Direct Binding Assays with Purified Proteins:

• Purify recombinant His-FAL1 and MBP-Sgd1-His

• Perform binding assays by immobilizing one protein and testing for interaction with the other

• Test the effect of ATP, non-hydrolyzable ATP analogs (ADPNP), and RNA on the interaction

Functional Assays:

• Test if the MIF4G domain of Sgd1 stimulates FAL1's ATPase activity in vitro

• Perform domain mapping to identify the regions essential for interaction

Research has demonstrated that FAL1 and Sgd1 form an RNA-independent interaction, as RNase treatment does not disrupt their association. Additionally, the interaction was not significantly affected by the presence of ATP, ADPNP, or RNA .

How can I design a study to investigate FAL1's role in ribosome biogenesis?

A comprehensive experimental design should include:

Genetic Approaches:

• Generate conditional FAL1 mutants (temperature-sensitive or depletion strains)

• Create catalytically inactive mutants (e.g., DQAD mutation)

• Complement FAL1-deficient strains with wild-type or mutant versions

Ribosome Analysis:

• Polysome profiling to assess ribosome assembly defects

• Northern blot analysis of rRNA processing intermediates

• Pulse-chase labeling to track rRNA maturation

Protein-RNA Interactions:

• RNA immunoprecipitation to identify FAL1-associated RNAs

• CRAC (Crosslinking and Analysis of cDNAs) to map FAL1 binding sites on pre-rRNAs

• In vivo protein-RNA crosslinking to determine binding sites on 18S pre-rRNA

Protein Interaction Studies:

• Affinity purification-mass spectrometry to identify FAL1-associated proteins

• Yeast two-hybrid screens to find direct interactors

• Co-localization studies to visualize FAL1 in pre-ribosomal complexes

Published work has established that FAL1's catalytic activity is required for SSU biogenesis and that it likely associates transiently with SSU processomes containing the AF Lcp5 .

How does FAL1 function compare between different Candida species?

While research on FAL1 across different Candida species is limited, comparative analysis reveals:

The study of FAL1 function across Candida species could provide insights into species-specific adaptations in ribosome biogenesis pathways and potentially reveal conserved mechanisms that could be targeted for antifungal development.

What is the relationship between FAL1 and drug resistance mechanisms in C. glabrata?

While FAL1 itself has not been directly implicated in drug resistance mechanisms, its essential role in ribosome biogenesis suggests potential indirect connections:

• Pleiotropic Drug Resistance Network: C. glabrata exhibits resistance to fluconazole through the pleiotropic drug response (PDR) network, primarily regulated by the transcription factor Pdr1. This network includes ATP-binding cassette transporters like CDR1 that function as drug efflux pumps .

• Translational Adaptation: As FAL1 is essential for ribosome biogenesis, it may indirectly influence how C. glabrata adapts to drug stress through translational reprogramming.

• Experimental Approach: To investigate potential connections, researchers could:

  • Analyze transcriptome changes in FAL1 mutants in response to antifungal stress

  • Investigate whether FAL1 expression is altered in drug-resistant clinical isolates

  • Test if modulation of FAL1 activity sensitizes resistant strains to antifungals

Research has shown that mutations in PDR1 are the most common cause of fluconazole resistance in clinical isolates, leading to hyperactivation of this transcription factor and upregulation of drug efflux pumps .

How can I design FAL1 mutants to dissect structure-function relationships?

Based on research with FAL1 and related DEAD-box helicases, the following approach is recommended:

Key Functional Domains for Mutation:

Experimental Validation Strategies:

• In vitro biochemical assays for ATP hydrolysis and RNA unwinding

• Yeast complementation tests to assess functional consequences in vivo

• Protein-protein interaction assays to determine effects on complex formation

Research has demonstrated that the DQAD mutation effectively abolishes FAL1's catalytic activity while the protein remains stable, making it an excellent tool for structure-function studies .

What are common challenges in purifying active recombinant FAL1 protein?

Researchers often encounter several challenges when purifying FAL1:

For optimal results with recombinant FAL1, both E. coli and baculovirus expression systems have been successfully used, with the choice depending on downstream applications and required protein quality .

How can I optimize the experimental design to study FAL1's role in C. glabrata pathogenesis?

When investigating FAL1's potential role in C. glabrata pathogenesis, consider this experimental framework:

Genetic Manipulation Strategies:

• Use conditional expression systems rather than deletion (as FAL1 is likely essential)

• Create hypomorphic alleles that maintain partial function

• Employ tissue-specific or infection-stage-specific promoters

In Vitro Infection Models:

• Macrophage infection assays (similar to those used for CgXbp1 studies)

• Epithelial cell adherence and invasion assays

• Biofilm formation assays with FAL1 mutants

In Vivo Models:

• Murine systemic infection models

• Galleria mellonella infection for initial screening

• Tissue-specific infection models (e.g., oral, vaginal)

Key Measurements:

• Fungal burden in tissues

• Host immune response markers

• Changes in FAL1 expression during infection progression

Research approaches similar to those used to study CgXbp1's role in macrophage infection could be adapted for FAL1 studies. For example, researchers could map genome-wide RNA polymerase II occupancy to delineate transcriptional responses during infection or stress conditions .