Inositol depletion restores vesicle transport in yeast phospholipid flippase mutants.

Kanako Yamagami,Takamitsu Sano,Shota Sakai,Tetsuo Mioka,Yasuyuki Igarashi,Kazuma Tanaka,Takaharu Yamamoto,Christopher Beh

doi:10.1371/journal.pone.0120108

Abstract

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.

Highlights

In eukaryotic cells, phospholipids are asymmetrically distributed across the plasma membrane bilayer; phosphatidylserine (PS) and phosphatidylethanolamine (PE) are enriched in the cytoplasmic leaflet, whereas phosphatidylcholine (PC) and sphingolipids are located in the exoplasmic leaflet
We found that ART5 overexpression even suppressed the growth defects of the Neo1p-depleted mutant, suppressing the growth defects of all the flippase mutants
Numerous gene-deletion mutants require inositol for growth [69, 70], presumably because (i) inositol is an essential precursor for the biosynthesis of PI and PI-derived molecules, (ii) inositol is involved in transcriptional regulation of numerous genes, and (iii) inositol depletion is a stress-inducing growth condition

Summary

Introduction

Phospholipids are asymmetrically distributed across the plasma membrane bilayer; phosphatidylserine (PS) and phosphatidylethanolamine (PE) are enriched in the cytoplasmic leaflet, whereas phosphatidylcholine (PC) and sphingolipids are located in the exoplasmic leaflet. Phospholipid flippases are type 4 P-type ATPases that translocate (‘flip’) phospholipids from the exoplasmic to the cytoplasmic leaflet, thereby generating and maintaining the phospholipid asymmetry of the plasma membrane and organellar membranes [1,2,3,4]. The yeast Saccharomyces cerevisiae encodes five flippases: Drs2p, Dnf1p, Dnf2p, Dnf3p, and Neo1p. Drs2p, Dnf3p, and Dnf1p/Dnf2p form complexes with non-catalytic subunits of the Cdc family: Cdc50p, Crf1p, and Lem3p, respectively. These interactions are required for the ER exit, proper localization, function, and activity of the flippases [11,12,13,14,15]. The defective drs2Δ, dnf1Δ dnf2Δ, and dnf3Δ mutants are phenocopied by cdc50Δ, lem3Δ, and crf1Δ mutants, respectively [11, 12]

Methods

Results

Conclusion