Takar M, Wu Y, Graham TR

Eukaryotic organisms typically express multiple type IV P-type ATPases (P4-ATPases), which establish plasma membrane asymmetry by flipping specific phospholipids from the exofacial to the cytosolic leaflet. Saccharomyces cerevisiae, for example, expresses five P4-ATPases including Neo1, Drs2, Dnf1, Dnf2 and Dnf3. Neo1 is thought to be a phospholipid flippase, although there ...

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is currently no experimental evidence that Neo1 catalyzes this activity or helps establish membrane asymmetry. Here we use temperature-conditional alleles (neo1(ts)) to test if Neo1 deficiency leads to loss of plasma membrane asymmetry. Wild-type (WT) yeast normally restrict most of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the inner, cytosolic leaflet of the plasma membrane. However, the neo1-1(ts) and neo1-2(ts) mutants display a loss of PS and PE asymmetry at permissive growth temperatures as measured by hypersensitivity to pore forming toxins that target PS (papuamide A) or PE (duramycin) exposed in the extracellular leaflet. When shifted to a semipermissive growth temperature, the neo1-2(ts) mutant became extremely hypersensitive to duramycin while sensitivity to papuamide A was unchanged, indicating preferential exposure of PE. This loss of asymmetry occurs in spite of the presence of other flippases that flip PS and/or PE. Even when overexpressed, Drs2 and Dnf1 were unable to correct the loss of asymmetry caused by neo1(ts) However, modest overexpression of Neo1 weakly suppressed loss of membrane asymmetry caused by drs2Δ with a more significant correction of PE asymmetry than PS. These results indicate that Neo1 plays an important role in establishing PS and PE plasma membrane asymmetry in budding yeast.

Date: May. 26, 2016

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