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GNPS Proximity labeling identification of plasma membrane eisosome proteins in Candida albicans

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 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. Two proteins that localize to these domains, Sur7 and Lsp1, are critical for virulence. Identifying additional proteins that localize to MCC/eisosomes has been hindered by the technical challenges of working with membrane proteins. Therefore, the proximity labeling method TurboID was used to identify MCC/eisosome proteins in C. albicans. Candidate proteins were identified by fusing a variant of BirA known as TurboID to either Sur7 or Lsp1, to promote biotinylation of nearby proteins. Analysis of 19 of these proteins by tagging with the Green Fluorescent Protein (GFP) 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 novel therapeutic strategies.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Candida Albicans (ncbitaxon:5476)

SUBMITTER: James B. Konopka

PROVIDER: MSV000097378 | GNPS | Wed Mar 19 10:51:00 GMT 2025

SECONDARY ACCESSION(S): PXD062040

REPOSITORIES: GNPS

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Project description:Topoisomerases are crucial to solve DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3M-NM-2 (Top3M-NM-2) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein deficient in Fragile X syndrome and known to regulate translation of mRNAs important for neuronal function and autism. Notably, the FMRP-Top3M-NM-2 interaction is abolished by a disease-associated FMRP mutation, and several human genetic studies link Top3M-NM-2 mutation to schizophrenia and intellectual disability. Top3M-NM-2 binds multiple mRNAs encoded by genes with neuronal functions related to schizophrenia and autism. Expression of one such gene, ptk2/FAK, is reduced in neuromuscular junctions of Top3M-NM-2 mutant flies. Synapse formation is defective in Top3M-NM-2 mutant flies and mice, as observed in FMRP mutant animals. Our findings suggest that Top3M-NM-2 acts as an RNA topoisomerase and works with FMRP to promote expression of mRNAs critical for neurodevelopment and mental health. We have identified Top3M-NM-2 as the first RNA topoisomerase in eukaryotes. To study whether Top3M-NM-2 binds mRNAs in vivo and also to identify its genome-wide RNA targets, we performed HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation; Licatalosi et al. Nature 456, 464-469, 2008). In this method, proteins are covalently crosslinked with bound RNAs in vivo, thus allowing specific mapping of the sites of interaction of the protein with the RNA. We cultured a HeLa cells stably-expressing HF-Top3M-NM-2 and irradiated the cells with UV to crosslink RNA and proteins in vivo. Following cell lysis, RNA was partially digested using various concentrations of RNase A. The RNA crosslinked to HF-Top3M-NM-2 was then immunoprecipitated with anti-Flag M2 argarose beads (Sigma). As a control, we also performed a mock immunoprecipitation using HeLa cells that do not express HF-Top3M-NM-2. The 3M-bM-^@M-^Y ends of the purified RNAs were ligated to a p32-labeled RNA adapter, fractionated by SDS-PAGE, and transferred to a nitrocellulose membrane. The p32-labeled RNA-Top3M-NM-2 complexes were detected by autoradiography and recovered from the membrane. The complexes were treated with Proteinase K to remove proteins. The RNA was ligated at their 5M-bM-^@M-^Yend to another RNA adaptor, and reverse-transcribed to cDNA. The cDNA obtained was fractionated a denaturing 6% TBE-Urea gel (InVitrogen), amplified by PCR, and subjected to high-throughput sequencing by Genome Analyzer II (Illumina). Sequenced tags were mapped to the human genome (hg18, NCBI/NIH) and to human RefSeq genes using ELAND program (Illumina).

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GNPS Proximity labeling identification of plasma membrane eisosome proteins in Candida albicans

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