P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity
Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen <i>Cryptococcus neoformans</i>, the P4-ATPase Apt1p is an important regulator of polysacch...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-10-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/10/843 |
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| author | Lyubomir Dimitrov Stanchev Juliana Rizzo Rebecca Peschel Lilli A. Pazurek Lasse Bredegaard Sarina Veit Sabine Laerbusch Marcio L. Rodrigues Rosa L. López-Marqués Thomas Günther Pomorski |
| author_facet | Lyubomir Dimitrov Stanchev Juliana Rizzo Rebecca Peschel Lilli A. Pazurek Lasse Bredegaard Sarina Veit Sabine Laerbusch Marcio L. Rodrigues Rosa L. López-Marqués Thomas Günther Pomorski |
| author_sort | Lyubomir Dimitrov Stanchev |
| collection | DOAJ |
| description | Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen <i>Cryptococcus neoformans</i>, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in <i>S. cerevisiae</i>, we demonstrate that Apt1p forms a heterodimeric complex with the <i>C. neoformans</i> Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in <i>C. neoformans</i> is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development. |
| first_indexed | 2024-03-10T06:27:51Z |
| format | Article |
| id | doaj.art-5d6bcff620d549f287656bfe50c80614 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T06:27:51Z |
| publishDate | 2021-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-5d6bcff620d549f287656bfe50c806142023-11-22T18:47:22ZengMDPI AGJournal of Fungi2309-608X2021-10-0171084310.3390/jof7100843P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate SpecificityLyubomir Dimitrov Stanchev0Juliana Rizzo1Rebecca Peschel2Lilli A. Pazurek3Lasse Bredegaard4Sarina Veit5Sabine Laerbusch6Marcio L. Rodrigues7Rosa L. López-Marqués8Thomas Günther Pomorski9Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyInstituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, BrazilDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyDepartment of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, DenmarkDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyInstituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, BrazilDepartment of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, DenmarkDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, GermanyLipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen <i>Cryptococcus neoformans</i>, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in <i>S. cerevisiae</i>, we demonstrate that Apt1p forms a heterodimeric complex with the <i>C. neoformans</i> Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in <i>C. neoformans</i> is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.https://www.mdpi.com/2309-608X/7/10/843β-subunitCDC50 proteinheterologous expressionlipid transportmembrane transport proteinP4-ATPase |
| spellingShingle | Lyubomir Dimitrov Stanchev Juliana Rizzo Rebecca Peschel Lilli A. Pazurek Lasse Bredegaard Sarina Veit Sabine Laerbusch Marcio L. Rodrigues Rosa L. López-Marqués Thomas Günther Pomorski P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity Journal of Fungi β-subunit CDC50 protein heterologous expression lipid transport membrane transport protein P4-ATPase |
| title | P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity |
| title_full | P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity |
| title_fullStr | P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity |
| title_full_unstemmed | P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity |
| title_short | P-Type ATPase Apt1 of the Fungal Pathogen <i>Cryptococcus neoformans</i> Is a Lipid Flippase of Broad Substrate Specificity |
| title_sort | p type atpase apt1 of the fungal pathogen i cryptococcus neoformans i is a lipid flippase of broad substrate specificity |
| topic | β-subunit CDC50 protein heterologous expression lipid transport membrane transport protein P4-ATPase |
| url | https://www.mdpi.com/2309-608X/7/10/843 |
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