Flippase

FUNCTIONAL ANALYSIS OF FLIPPASES IN YEAST

1. Functional analysis of the flippases i Qussai Z Ph.D. Microbiology

2. OUTLINES INTRODUCTION PROBLEM STATEMENT JUSTIFICATION OBJECTIVE METHODOLOGY CURRENT RESULTS

3. P4-ATPases (Flippases) are unique to eukaryotes, where they are present in the plasma membrane and membranes of the secretory pathway. And is essential for maintaining membrane lipid asymmetry, signaling, vesicle formation, and regulation of membrane protein activity. The term ‘ flippase ’ refer to the bidirectional lipid transporters. Perez et al., 2015 doi:10.1038/nature14953

4. So how does Flippase work?   The flippase homodimer has three main structural features: a nucleotide binding site, a positively charged pocket within the membrane-spanning helices, and an external helix.  Perez et al., 2015 doi:10.1038/nature14953

5. The P4-ATPases subfamily comprises five members in yeast (Drs2, Dnf1, Dnf2, Dnf3 and Neo1)  The active unit of flippases is a heterodimeric αβ complex: while the P4-ATPase constitutes the catalytic α- subunit, the β- subunit exhibits chaperone-like features and belongs to the Cdc50 family. The P4-ATPases present in Saccharomyces cerevisiae are well characterized, and are involved in phospholipid translocation and vesiculating. In total, S. cerevisiae  harbours five members of this family, namely Neo1p (Neomycin resistant 1), Drs2p (Deficient for Ribosomal Subunit 2), Dnf1p (Drs2p/Neo1p family), Dnf2p and Dnf3p. Neo1p is the only essential member of the family while the four remaining members can be knocked out in all combinations, except for all four at once, without leading to lethality.

6. Substrate specificities and biological roles of flippases. Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032

7. Flippases participate in key biological processes: Vesicular trafficking Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032

8. Potential participation of components of the endosomal sorting complex required for transport (ESCRT) machinery, GRASP and Flippases in the biogenesis of fungal extracellular vesicles Oliveira et al., 2013 ,  14 (5), 9581-9603; doi: 10.3390/ijms14059581

9. Regulation of the trans-bilayer lipid distribution in cellular membranes. Thomas Pomorski et al. J Cell Sci 2004;117:805-813 © The Company of Biologists Limited 2004

10. Role of energy-coupled lipid flippases in triggering membrane budding. Thomas Pomorski et al. J Cell Sci 2004;117:805-813 © The Company of Biologists Limited 2004

11. The transport cycle of flippases and Na+/K+-pumps. Flippases evolved from a family of cation pumps Radhakrishnan Panatala et al. J Cell Sci 2015;128:2021-2032

12. Experimental procedures Fungal strains, media, and culture conditions Constructs for gene deletion and complementation mutants Construction of GFP and RFP fusion cassettes Yeast two‑hybrid analyses Split‑ubiquitin yeast two‑hybrid (Y2H) assay Microscopic examinations of hyphal and conidial morphology Growth, conidiation, and stress‑sensitivity tests Virulence assay Western blot analysis Affinity capture–mass spectrometry analysis Co‑immunoprecipitation assay