Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

Abstract

Lem3p-Dnf1p is a putative aminophospholipid translocase (APLT) complex that is localized to the plasma membrane; Lem3p is required for Dnf1p localization to the plasma membrane. We have identified lem3 mutations, which did not affect formation or localization of the Lem3p-Dnf1p complex, but caused a synthetic growth defect with the null mutation of CDC50, a structurally and functionally redundant homologue of LEM3. Interestingly, these lem3 mutants exhibited nearly normal levels of NBDlabeled phospholipid internalization across the plasma membrane, suggesting that Lem3p may have other functions in addition to regulation of the putative APLT activity of Dnf1p at the plasma membrane. Similarly, deletion of the COOH-terminal cytoplasmic region of Dnf1p affected neither the localization nor the APLT activity of Dnf1p at the plasma membrane, but caused a growth defect in the cdc50∆ background.Our results suggest that the Lem3p-Dnf1p complex may play a role distinct from its plasma membrane APLT activity when it substitutes for the Cdc50p-Drs2p complex, its redundant partner in the endosomal/trans-Golgi network compartments.3

A novel factorOPT2mediates exposure of phospholipids during cellular adaptation to altered lipid asymmetry

“…5B). The structure of Ro-09-198 closely resembles that of duramycin (Noji et al, 2006). As described in the literature (Iwamoto et al, 2004;Saito et al, 2007), PtdEtn was highly exposed in lem3D cells but little PtdEtn was exposed in wild-type cells.…”

“…In lem3D cells, PtdEtn and PtdSer, normally confined to the inner leaflet of the plasma membrane, are exposed to the cell surface (the outer leaflet) owing to the defective flippase activity; as a result, lem3D cells were hypersensitive to both duramycin and papuamide B (Fig. 5A) (Noji et al, 2006;Parsons et al, 2006). Deletion of either OPT2 or RIM21 in lem3D cells (opt2D lem3D or rim21D lem3D cells, respectively) conferred some Fig.…”

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity

“…29 Drs2p assembles with Cdc50p and cycles between the trans-Golgi network (TGN), late endosomes, and the plasma membrane. 3,17,18 Dnf1p and Dnf2p interact with Lem3p 17,19 and cycle between the endosomal system and the plasma membrane. 3,4 Finally, Neo1p cycles between ER and TGN and TGN and the endosomal system, 31,32 whereas Dnf3p has overlapping localizations with Drs2p; 3,4 however, no assembly with Cdc50 proteins has been reported thus far.…”

“…[17][18][19][20] In yeast, this evolutionary conserved family of transmembrane proteins includes three close Cdc50p homologs, which are glycosylated proteins of approximately 60 kDa with two putative transmembrane domains. Saito and colleagues 17 were the first to show that in yeast interactions between P4 ATPases and Cdc50p are required for the release of P4 ATPases from the endoplasmic reticulum (ER) and targeting to their proper membrane domains.…”

Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry

“…Additionally, we showed that a complex sphingolipid species, mannosyl-inositol-phosphorylceramide (MIPC), is required for Fpk1 activity (Roelants et al, 2010). Although these observations uncovered novel inputs into Fpk1 regulation, they did not explain how Fpk1-dependent regulation of flippases is controlled spatially and temporally during passage escort protein, Lem3/Ros3 (414 residues; Kato et al, 2002;Noji et al, 2006). Genetic analysis in yeast has implicated Dnf1 and Dnf2, and the other flippases-and thus membrane asymmetry-in endocytosis, protein trafficking, and vesicle formation (Chen et al, 1999;Gall et al, 2002;Hua et al, 2002;Pomorski et al, 2003;Liu et al, 2007;Natarajan et al, 2009;Hachiro et al, 2013) and in establishment of cell polarity (Iwamoto et al, 2004;Saito et al, 2007;Fairn et al, 2011;Das et al, 2012).…”