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Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis
Published on: June 13, 2021
Proximity labeling identification of plasma membrane eisosome proteins in Candida albicans
Carla E Lanze1, John D Haley2,3, James B Konopka1
1Department of Microbiology and Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Abstract:
The ability of Candida albicans to resist stressful conditions in the host and grow invasively into tissues contributes to the virulence of this human fungal pathogen. Plasma membrane subdomains known as the MCC (membrane compartment of Can1) or eisosomes are important for these processes. MCC/eisosome domains are furrow-shaped invaginations of the plasma membrane that are about 250 nm long and 50 nm deep. To identify proteins that localize to these domains, a proximity labeling method was used in which the TurboID variant of the BirA biotin ligase was fused to Sur7 and Lsp1, 2 proteins that localize to eisosomes and are important for virulence. This resulted in biotinylation of nearby proteins, permitting their identification. Analysis of 19 candidate proteins by tagging with the green fluorescent protein identified 7 proteins that detectably overlapped with MCC/eisosomes. Deletion mutant analysis showed that one of these, a poorly studied protein known as Ker1, was important for hyphal growth in liquid culture, invasive growth into agar medium, and resistance to stress caused by copper and cell wall perturbing agents. Altogether, these approaches identified novel MCC/eisosome proteins and show that TurboID can be applied to better define the molecular mechanisms of C. albicans pathogenesis and aid in discovery of targets for novel therapeutic strategies.
Insights
Researchers identified new proteins in Candida albicans's membrane compartments (MCCs/eisosomes) using proximity labeling. One protein, Ker1, is crucial for fungal virulence and stress resistance, offering potential therapeutic targets.
Area of Science:
- Mycology
- Cell Biology
- Pathogenesis
Background:
- Candida albicans virulence relies on its ability to withstand host stress and invade tissues.
- Plasma membrane subdomains, MCCs (membrane compartment of Can1) or eisosomes, are critical for these pathogenic processes.
- MCCs/eisosomes are distinct, furrow-shaped invaginations of the plasma membrane.
Purpose of the Study:
- To identify novel proteins localizing to MCC/eisosome domains in Candida albicans.
- To investigate the role of identified proteins in fungal virulence and stress response.
- To evaluate the utility of TurboID proximity labeling for studying C. albicans pathogenesis.
Main Methods:
- Proximity labeling using TurboID fused to known eisosome proteins (Sur7, Lsp1).
- Biotinylation and identification of nearby proteins.
- Green fluorescent protein tagging for protein localization analysis.
- Deletion mutant analysis to assess protein function.
Main Results:
- Successfully identified 7 novel proteins localizing to MCC/eisosomes out of 19 candidates.
- Ker1, a previously understudied protein, was confirmed to localize to MCC/eisosomes.
- Ker1 deletion mutants exhibited defects in hyphal growth, invasive growth, and stress resistance.
Conclusions:
- Novel MCC/eisosome proteins were identified, expanding the understanding of these domains.
- Ker1 plays a significant role in Candida albicans virulence and stress adaptation.
- TurboID is a valuable tool for dissecting C. albicans pathogenesis and identifying therapeutic targets.
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