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Updated: Oct 16, 2025

A Fluorescence-based Assay of Phospholipid Scramblase Activity
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P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

Lyubomir Dimitrov Stanchev1,2, Juliana Rizzo3,4, Rebecca Peschel1

  • 1Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, 44780 Bochum, Germany.

Journal of Fungi (Basel, Switzerland)
|October 23, 2021
PubMed

Insights

The P4-ATPase Apt1p in Cryptococcus neoformans forms a complex with Cdc50, enabling lipid transport essential for fungal virulence. This finding highlights Apt1p as a potential target for developing new antifungal drugs.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Mycology

Background:

  • P4-ATPases are ATP-driven lipid transporters crucial for membrane asymmetry.
  • Apt1p, a P4-ATPase in the fungal pathogen Cryptococcus neoformans, is implicated in pathogenesis but lacks biochemical characterization.
  • Apt1p belongs to a P4A-ATPase subclade requiring a beta-subunit.

Purpose of the Study:

  • To biochemically characterize the P4-ATPase Apt1p from Cryptococcus neoformans.
  • To investigate the role of the Cdc50 protein in Apt1p function and localization.
  • To determine the substrate specificity of the Apt1p-Cdc50 complex.

Main Methods:

  • Phylogenetic analysis of P4-ATPases.
  • Heterologous expression of Apt1p and Cdc50 in Saccharomyces cerevisiae.
  • Complementation tests and fluorescent lipid uptake assays to assess flippase activity.

Main Results:

  • Apt1p forms a functional heterodimeric complex with Cdc50, essential for its localization and activity.
  • The Apt1p-Cdc50 complex exhibits broad substrate specificity, transporting phospholipids, miltefosine, and glycolipids.
  • Lipid transport by Apt1p is crucial for Cryptococcus neoformans virulence.

Conclusions:

  • The Apt1p-Cdc50 complex is a key regulator of transbilayer lipid transport in Cryptococcus neoformans.
  • Fine-tuned lipid transport by Apt1p contributes to fungal pathogenesis.
  • Apt1p represents a promising target for novel antifungal drug development.
Keywords:
CDC50 proteinP4-ATPaseheterologous expressionlipid transportmembrane transport proteinβ-subunit

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