DeMarco, A. G. | Search bioRxiv

bioRxiv2023

Inducible degradation-coupled phosphoproteomics identifies PP2A Rts1 as a novel eisosome regulator

Reversible protein phosphorylation is an abundant post-translational modification dynamically regulated by opposing kinases and phosphatases. Protein phosphorylation has been extensively studied in cell division, where waves of cyclin-dependent kinase activity, peaking in mitosis, drive the sequential stages of the cell cycle. Here we developed and employed a strategy to specifically probe kinase or phosphatase substrates at desired times or experimental conditions in the model organism Saccharomyces cerevisiae. We combined auxin- inducible degradation (AID) with mass spectrometry-based phosphoproteomics, which allowed us to arrest physiologically normal cultures in mitosis prior to rapid phosphatase degradation and phosphoproteome analysis. Our results revealed that protein phosphatase 2A coupled with its B56 regulatory subunit, Rts1 (PP2ARts1), is involved in dephosphorylation of numerous proteins in mitosis, highlighting the need for phosphatases to selectively maintain certain proteins in a hypophosphorylated state in the face of high mitotic kinase activity. Unexpectedly, we observed elevated phosphorylation at many sites on several subunits of the fungal eisosome complex following rapid Rts1 degradation. Eisosomes are dynamic polymeric assemblies that create furrows in the plasma membrane important in regulating nutrient import, lipid metabolism, and stress responses, among other things. We found that PP2ARts1-mediated dephosphorylation of eisosomes promotes their plasma membrane association and we provide evidence that this regulation impacts eisosome roles in metabolic homeostasis. The combination of rapid, inducible protein degradation with proteomic profiling offers several advantages over common protein disruption methods for characterizing substrates of regulatory enzymes involved in dynamic biological processes.

molecular biology↗

bioRxiv2022

Reduced Cdc14 phosphatase activity impairs septation, hyphal differentiation and pathogenesis and causes echinocandin hypersensitivity in Candida albicans

The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires a small fraction of normal Cdc14 activity. It remains unclear what fungal Cdc14 functions require high Cdc14 activity. We identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm) as the sole source of Cdc14 exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reducing Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity promotes fungal cell wall integrity and, in C. albicans, is needed to orchestrate septation and hyphal differentiation, and for pathogenesis. Cdc14 may therefore be worth future exploration as an antifungal drug target. AUTHOR SUMMARYInvasive fungal infections are a serious concern for the immune-compromised. Antifungal drugs to treat invasive infections are limited and pathogens are developing resistance to them. Novel targets for antifungal drug development are needed. In this study we developed a system to test if partial therapeutic reduction in activity of a protein phosphatase called Cdc14 could reduce virulence of the opportunistic human pathogen Candida albicans. This idea arose from prior studies in fungal pathogens of plants, where Cdc14 was unexpectedly required for host infection through an unknown mechanism. We found that successful C. albicans infections in two animal models of invasive candidiasis were dependent on high Cdc14 activity. Moreover, we made the surprising observation that integrity of the C. albicans cell wall is also dependent on high Cdc14 activity, with Cdc14-deficient cells becoming hypersensitive to cell wall-targeted antifungal drugs. We conclude that even modest reduction in Cdc14 activity could have therapeutic benefit for human fungal infections and possibly help overcome resistance to some antifungal drugs. Cdc14 structure and specificity are unique among phosphatases and highly conserved in pathogenic fungi, suggesting that highly selective inhibitors can be developed that would be useful against a broad range of fungal pathogens.

molecular biology↗

bioRxiv2020

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipid-accessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino- and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility.

biophysics↗