Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i>
Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fun...
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| Format: | Article |
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MDPI AG
2023-02-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/9/2/202 |
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| author | Sarina Veit Sabine Laerbusch Rosa L. López-Marqués Thomas Günther Pomorski |
| author_facet | Sarina Veit Sabine Laerbusch Rosa L. López-Marqués Thomas Günther Pomorski |
| author_sort | Sarina Veit |
| collection | DOAJ |
| description | Lipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen <i>Cryptococcus neoformans</i> contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient <i>S. cerevisiae</i> strain <i>dnf1Δdnf2Δdrs2Δ</i>, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the <i>C. neoformans</i> Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and –choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of <i>dnf1Δdnf2Δdrs2Δ</i>, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a β-subunit. These results identify <i>C. neoformans</i> Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions. |
| first_indexed | 2024-03-11T08:35:52Z |
| format | Article |
| id | doaj.art-92e2afa9ae694676a60756172a3a024e |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-11T08:35:52Z |
| publishDate | 2023-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-92e2afa9ae694676a60756172a3a024e2023-11-16T21:30:30ZengMDPI AGJournal of Fungi2309-608X2023-02-019220210.3390/jof9020202Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i>Sarina Veit0Sabine Laerbusch1Rosa L. López-Marqués2Thomas Günther Pomorski3Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, GermanyDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, GermanyDepartment of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, DenmarkDepartment of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44801 Bochum, GermanyLipid flippases of the P4-ATPase family actively transport phospholipids across cell membranes, an activity essential for key cellular processes such as vesicle budding and membrane trafficking. Members of this transporter family have also been implicated in the development of drug resistance in fungi. The encapsulated fungal pathogen <i>Cryptococcus neoformans</i> contains four P4-ATPases, among which Apt2-4p are poorly characterized. Using heterologous expression in the flippase-deficient <i>S. cerevisiae</i> strain <i>dnf1Δdnf2Δdrs2Δ</i>, we tested their lipid flippase activity in comparison to Apt1p using complementation tests and fluorescent lipid uptake assays. Apt2p and Apt3p required the co-expression of the <i>C. neoformans</i> Cdc50 protein for activity. Apt2p/Cdc50p displayed a narrow substrate specificity, limited to phosphatidylethanolamine and –choline. Despite its inability to transport fluorescent lipids, the Apt3p/Cdc50p complex still rescued the cold-sensitive phenotype of <i>dnf1Δdnf2Δdrs2Δ</i>, suggesting a functional role for the flippase in the secretory pathway. Apt4p, the closest homolog to Saccharomyces Neo1p, which does not require a Cdc50 protein, was unable to complement several flippase-deficient mutant phenotypes, neither in the presence nor absence of a β-subunit. These results identify <i>C. neoformans</i> Cdc50 as an essential subunit for Apt1-3p and provide a first insight into the molecular mechanisms underlying their physiological functions.https://www.mdpi.com/2309-608X/9/2/202β-subunitCDC50 proteinfunctional complementationheterologous expressionlipid transportlipid flippase |
| spellingShingle | Sarina Veit Sabine Laerbusch Rosa L. López-Marqués Thomas Günther Pomorski Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> Journal of Fungi β-subunit CDC50 protein functional complementation heterologous expression lipid transport lipid flippase |
| title | Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> |
| title_full | Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> |
| title_fullStr | Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> |
| title_full_unstemmed | Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> |
| title_short | Functional Analysis of the P-Type ATPases Apt2-4 from <i>Cryptococcus neoformans</i> by Heterologous Expression in <i>Saccharomyces cerevisiae</i> |
| title_sort | functional analysis of the p type atpases apt2 4 from i cryptococcus neoformans i by heterologous expression in i saccharomyces cerevisiae i |
| topic | β-subunit CDC50 protein functional complementation heterologous expression lipid transport lipid flippase |
| url | https://www.mdpi.com/2309-608X/9/2/202 |
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