Membrane homeostasis beyond fluidity: control of membrane compressibility

Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility

Mechanism of lipid droplet formation by the yeast Sei1/Ldb16 Seipin complex

Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity

Water soluble lipid precursor contaminants in yeast culture medium ingredients

The presence of the water soluble glycerophospholipid precursors choline and inositol in culture media highly affects lipid biosynthesis and regulation thereof. We report that widely used media ingredients contain trace amounts of choline and inositol that are not mentioned on the product label, influencing experimental outcome

The role of phospholipid molecular species in determining the physical properties of yeast membranes

In most eukaryotes, including Saccharomyces cerevisiae, glycerophospholipids are the main membrane lipid constituents. Besides serving as general membrane 'building blocks', glycerophospholipids play an important role in determining the physical properties of the membrane, which are crucial for proper membrane function. To ensure optimal physical properties, membrane glycerophospholipid composition and synthesis are tightly regulated. This review will summarize our current knowledge of factors and processes determining the membrane glycerophospholipid composition of the reference eukaryote S. cerevisiae at the level of molecular species. Extrapolating from relevant model membrane data, we also discuss how modulation of the molecular species composition can regulate membrane physical properties

The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis

Lipid Acyl Chain Remodeling in Yeast

Membrane lipid homeostasis is maintained by de novo synthesis, intracellular transport, remodeling, and degradation of lipid molecules. Glycerophospholipids, the most abundant structural component of eukaryotic membranes, are subject to acyl chain remodeling, which is defined as the post-synthetic process in which one or both acyl chains are exchanged. Here, we review studies addressing acyl chain remodeling of membrane glycerophospholipids in Saccharomyces cerevisiae, a model organism that has been successfully used to investigate lipid synthesis and its regulation. Experimental evidence for the occurrence of phospholipid acyl chain exchange in cardiolipin, phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine is summarized, including methods and tools that have been used for detecting remodeling. Progress in the identification of the enzymes involved is reported, and putative functions of acyl chain remodeling in yeast are discussed

Water soluble lipid precursor contaminants in yeast culture medium ingredients

The presence of the water soluble glycerophospholipid precursors choline and inositol in culture media highly affects lipid biosynthesis and regulation thereof. We report that widely used media ingredients contain trace amounts of choline and inositol that are not mentioned on the product label, influencing experimental outcome

The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis