Phosphatidylserine prevents the generation of a protein-free giant plasma membrane domain in yeast

Abstract

Membrane phase separation accompanied with micron-scale domains of lipids and proteins occurs in artificial membranes; however, a similar large phase separation has not been reported in the plasma membrane of the living cells. We demonstrate here that a stable micron-scale protein-free region is generated in the plasma membrane of the yeast mutants lacking phosphatidylserine. We named this region the ″void zone″. Transmembrane proteins, peripheral membrane proteins, and certain phospholipids are excluded from the void zone. The void zone is rich in ergosterol and requires ergosterol and sphingolipids for its formation. These characteristics of the void zone are similar to the properties of the liquid-ordered domain caused by phase separation. We propose that phosphatidylserine prevents the formation of the void zone by preferentially interacting with ergosterol. We also found that void zones were frequently in contact with vacuoles, in which a membrane domain was also formed at the contact site.

Let it stick: Strategies and applications for intracellular plasma membrane targeting of proteins inSaccharomyces cerevisiae

“…In mammalian cells, using a tandem PH Osh2p (GFP-2xPH Osh2p ) led to a decrease in nuclear and an increase PM localization in contrast to the solo domain (Balla et al, 2008). In yeast, Mioka et al (2020) showed that the tandem of PH Osh2p (2xPH Osh2p -3xGFP) also targeted to the PM. Roy and Levine (2004) (Yu et al, 2004).…”

“…Multiplex lipid binding domains can appear in homo‐tandems of PH domains such as in mammalian TAPP1 and TAPP2 (Li et al, 2013), as hetero‐tandems with other lipid binding domains such as the tandem PX‐PH domains in yeast Bem3p that cooperatively bind membrane lipids (Ali et al, 2018) or a combination of tandem PH domains and additional motifs such as for human ARAP3 (Craig et al, 2010). Tandem configurations of PH domains that appear naturally as single domains were artificially created for rat and human PH PLCδ1 (GFP‐2xPH PLCδ1 ) and tested in yeast (Fairn et al, 2011; Mioka et al, 2020; Omnus et al, 2018; Rodríguez‐Escudero et al, 2018; Thomas et al, 2022; Wild et al, 2004). In direct comparison to GFP‐PH PLCδ1 , GFP‐2xPH PLCδ1 showed that previously weak PM binding with high levels of cytosolic localization was shifted to strong PM targeting in yeast (Levine & Munro, 2002; Wild et al, 2004).…”

“…In mammalian cells, using a tandem PH Osh2p (GFP‐2xPH Osh2p ) led to a decrease in nuclear and an increase PM localization in contrast to the solo domain (Balla et al, 2008). In yeast, Mioka et al (2020) showed that the tandem of PH Osh2p (2xPH Osh2p ‐3xGFP) also targeted to the PM. Roy and Levine (2004) used a tandem of PH Osh2p flanked by GFP on each site and saw that the fluorescent probe not only localized to punctuate internal structures as the monomer but also to the PM.…”