The plasma membrane delimits the cell, and its integrity is essential for cell survival. Lipids and proteins form domains of distinct composition within the plasma membrane. How changes in plasma membrane composition are perceived, and how the abundance of lipids in the plasma membrane is regulated to balance changing needs remains largely unknown. Here, we show that the Slm1/2 paralogues and the target of rapamycin kinase complex 2 (TORC2) play a central role in this regulation. Membrane stress, induced by either inhibition of sphingolipid metabolism or by mechanically stretching the plasma membrane, redistributes Slm proteins between distinct plasma membrane domains. This increases Slm protein association with and activation of TORC2, which is restricted to the domain known as the membrane compartment containing TORC2 (MCT; ref. ). As TORC2 regulates sphingolipid metabolism, our discoveries reveal a homeostasis mechanism in which TORC2 responds to plasma membrane stress to mediate compensatory changes in cellular lipid synthesis and hence modulates the composition of the plasma membrane. The components of this pathway and their involvement in signalling after membrane stretch are evolutionarily conserved.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl‐inositol‐anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha‐factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII‐coated, ER‐derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER‐derived transport vesicles.

In Saccharomyces cerevisiae, alpha‐factor is internalized by receptor‐mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta‐tubulin and actin mutant strains, we showed that actin plays a direct role in receptor‐mediated internalization of alpha‐factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta‐tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin‐binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha‐factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.

Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS)) encoding the 125-kDa protein (Gaslp) and found that the function of Gaslp is not essential for cell viability. The nucleotide sequence of GAS) predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N-and 0-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gaslp is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gaslp depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.A number of eucaryotic membrane proteins are anchored to the lipid bilayer by a covalently linked glycosyl phosphatidylinositol (GPI) moiety (reviewed in references 20, 26, and 46). This particular mode of membrane attachment occurs in a wide variety of eucaryotic organisms. The modified proteins fall into diverse functional groups, including hydrolytic enzymes, cell adhesion molecules, protozoan coat proteins, and numerous cell surface antigens of unknown function.The complete structure of the GPI moiety has been determined for two forms of the variant surface glycoprotein of Trypanosoma brucei (25, 58) and the mammalian cell surface antigen . GPI anchors from these distantly related organisms share a common core structure, consisting of a phosphatidylinositol molecule linked to a linear tetrasaccharide composed of one nonacetylated glucosaminyl and three mannosyl residues. At its nonreducing end, the glycan is attached via a phosphodiester to ethanolamine, which is amide linked to the a-carboxyl group of the C-terminal amino acid of the mature protein.GPI-anchored proteins are commonly synthesized with a cleavable N-terminal signal sequence and a C-terminal domain composed predominantly of hydrophobic amino acids. This particular feature seems to be important in the mechanism of anchor addition. In all cases studied so far, addition of the GPI anchor involves the removal of 17 to 31 residues from the C terminus of a larger precursor (7,15,22,27,29,32,35,36,47,51,53,60,62,65,68). Since processing rapidly follows protein synthesis (2, 18, 24), it is believed that the GPI moiety is preassembled and transferred en bloc to the protein in the endoplasmic reticulum.Several lines of evidence suggest that a signal for GPI anchor attachment resides in the C-terminal domain of the proteins. However, the C-terminal sequences of GPI-anchored proteins do not exhibit any recognizable homology. Although the stru...

Oxygen deprivation is rapidly deleterious for most organisms. However, Caenorhabditis elegans has developed the ability to survive anoxia for at least 48 hours. Mutations in the DAF-2/DAF-16 insulin-like signaling pathway promote such survival. We describe a pathway involving the HYL-2 ceramide synthase that acts independently of DAF-2. Loss of the ceramide synthase gene hyl-2 results in increased sensitivity of C. elegans to anoxia. C. elegans has two ceramide synthases, hyl-1 and hyl-2, that participate in ceramide biogenesis and affect its ability to survive anoxic conditions. In contrast to hyl-2(lf) mutants, hyl-1(lf) mutants are more resistant to anoxia than normal animals. HYL-1 and HYL-2 have complementary specificities for fatty acyl chains. These data indicate that specific ceramides produced by HYL-2 confer resistance to anoxia.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)‐anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide‐containing, mild base‐resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI‐anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI‐anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI‐anchored proteins.

The ER–mitochondrial encounter structure (ERMES) physically links ER and mitochondrial membranes in yeast, but it is unclear whether ERMES directly facilitates lipid exchange between these organelles. Kawano et al. reveal by reconstitution experiments that a complex of Mmm1–Mdm12, two core subunits of ERMES, functions as a minimal unit for lipid transfer between membranes.