Phospholipid flippases and Sfk1 are essential for the retention of ergosterol in the plasma membrane

Abstract

Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

Tricalbin proteins regulate plasma membrane phospholipid homeostasis

“…In reciprocal experiments, we addressed whether the Tcb proteins also regulate sterol availability at the PM (Fig 4F). In wild-type cells, the sterol probe mCherry-D4H (Maekawa & Fairn, 2015;Marek et al, 2020) localised primarily to the daughter bud with relatively low levels observed at the PM of the mother cell (Fig 4F ), consistent with previous reports (Marek et al, 2020;Kishimoto et al, 2021). However, the distribution of the sterol probe was altered in cells lacking the Tcb proteins, with increased mCherry-D4H signal at the PM of the mother cell (~3-fold greater than wild type control cells; Fig 4F).…”

“…The data also show that the Tcb proteins regulate phospholipid homeostasis at the PM upon stress conditions. Furthermore, we find that Tcb3 co-localises with the PM protein Sfk1, an ortholog of the mammalian TMEM150/FRAG1/DRAM proteins, that is implicated in stress-induced PI(4,5)P 2 synthesis and phospholipid asymmetry at the PM (Audhya & Emr, 2002;Chung et al, 2015;Mioka et al, 2018;Kishimoto et al, 2021). Finally, we find that the Tcb proteins promote the recruitment of Pkc1 to the mother cell cortex upon heat stress conditions.…”

“…However, PI4P 5-kinases are recruited and activated by PS (Fairn et al, 2009;Nishimura et al, 2019), and regulation of PS by Sfk1 and the Tcb proteins may indirectly regulate PI(4,5)P 2 synthesis at the PM. Interestingly, Sfk1 is also implicated in PM phospholipid asymmetry and PM integrity (Mioka et al, 2018;Kishimoto et al, 2021). As PI(4,5)P 2 synthesis is required for PM integrity (Audhya et al, 2000;Omnus et al, 2016), one possibility is that Tcb3 and Sfk1 promote the formation of PS-enriched domains that promote PI4P 5-kinase activity upon PM stress.…”

“…ER-localised Tcb3 associates with Sfk1 at the PM Sfk1 is an integral PM protein that has been implicated in PM phospholipid asymmetry and PM integrity (Mioka et al, 2018;Kishimoto et al, 2021), as well as heat-induced PI(4,5)P 2 synthesis (Audhya & Emr, 2002). We observed that the ER-localised Tcb3 protein is in close proximity to the integral PM protein As mentioned, Sfk1 has been implicated in PM lipid organisation and integrity (Mioka et al, 2018;Kishimoto et al, 2021). Tcb3 has also been shown to be necessary for maintenance of PM integrity upon heat stress conditions (Collado et al, 2019).…”

“…Accordingly, PM localisation of the PS FLARE was decreased upon a brief shift to 42°C upon loss of Sfk1 or truncation of the Sfk1 cytoplasmic tail (Fig 4E ), similar to tcb1/2/3Δ mutant cells (Fig 3C). Sfk1 has recently been shown to regulate PM sterol organisation (Kishimoto et al, 2021). In reciprocal experiments, we addressed whether the Tcb proteins also regulate sterol availability at the PM (Fig 4F).…”