Proteomic and bioinformatic analysis of outer membrane proteins of the protobacterium Bartonella henselae (Bartonellaceae)

Bartonella henselae, an infectious agent causing cat-scratch disease and vasculoproliferative disorders in humans, is a fastidious facultative intracellular pathogen. The outer membrane proteins of B. henselae are key molecules that play a primary role in host-cell interactions. We isolated B. henselae outer membrane proteins, using the ionic detergent N-lauroyl sarcosine sodium salt and sodium carbonate, purification by two-dimensional (2-D) gel electrophoresis, and protein identification using mass spectrometry. Treatment with buffers containing ASB-14 and ZWITTERGENT 3-10 increased solubilization of B. henselae proteins, particularly proteins with basic pI. Three hundred and sixty-eight spots were detected from the sarcosine-insoluble outer membrane fraction; 94 distinct protein species were identified from 176 spots. In the outer membrane fraction from carbonate incubation, 471 spots were calculated and 259 spots were identified, which included 139 protein entries. There were six outer membrane proteins in the sarcosine-insoluble outer membrane fraction compared with nine outer membrane proteins from samples subjected to carbonate incubation. We used bioinformatic analysis to identify 44 outer membrane proteins by prediction of their domains and tertiary structures and documented the potential virulence factors. We established the 2-D reference maps of the outer membrane subproteome of B. henselae using the two different extraction methods, which were partly complementary to each other. Sodium carbonate extraction isolated low-abundance and basic proteins better than the lauroyl sarcosine sodium salt extraction, which enriched high-abundance porins.     

Proteomic analysis of a membrane skeleton fraction from human liver

The cytoskeleton networks around liver cell cortex can resist Triton extraction and co-pellet with their tightly associated integral membrane proteins, forming assemblies called "membrane skeletons". Despite their important roles in determining cell shape and in signal transduction pathways, the membrane skeletons of human liver cells are uncharacterized to a great extent. In the present work, we prepared a membrane skeleton fraction by Triton extraction of human liver plasma membranes and then separated its protein components by 2-D gels. We optimized the detergent used for protein solubilization and found that 2% ASB-14 allowed the best recovery of membrane skeleton proteins. By analyzing the protein spots with MALDI-TOF and MALDI-TOF-TOF MS, we identified 104 nonredundant proteins, wherein 38 were cytoskeletal proteins that were further classified into several groups, including proteins in fodrin-based meshworks, adhesion proteins (proteins involved in adherens junctions, focal adhesions, desmosomes, hemidesmosomes and tight junctions), proteins that regulate F-actin dynamics, motor proteins, and some other cytoskeletal proteins. To the best of our knowledge, this is one of the largest data sets of membrane skeleton proteins to date. All the results suggested that the liver cells had complex actin- and cytokeratin-based membrane skeletons. This work provided a representative 2-DE map of membrane skeletons from human normal liver, for the purpose of helping to elucidate the composition and function of the membrane skeletons.     

Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the and subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75–5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.     

Domain-specific lipid distribution in macrophage plasma membranes

Lipid rafts, defined as cholesterol- and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques.     

Proteomic signatures for histological types of lung cancer

We performed proteomic studies on lung cancer cells to elucidate the mechanisms that determine histological phenotype. Thirty lung cancer cell lines with three different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma and adenocarcinoma) were subjected to two-dimensional difference gel electrophoresis (2-D DIGE) and grouped by multivariate analyses on the basis of their protein expression profiles. 2-D DIGE achieves more accurate quantification of protein expression by using highly sensitive fluorescence dyes to label the cysteine residues of proteins prior to two-dimensional polyacrylamide gel electrophoresis. We found that hierarchical clustering analysis and principal component analysis divided the cell lines according to their original histology. Spot ranking analysis using a support vector machine algorithm and unsupervised classification methods identified 32 protein spots essential for the classification. The proteins corresponding to the spots were identified by mass spectrometry. Next, lung cancer cells isolated from tumor tissue by laser microdissection were classified on the basis of the expression pattern of these 32 protein spots. Based on the expression profile of the 32 spots, the isolated cancer cells were categorized into three histological groups: the squamous cell carcinoma group, the adenocarcinoma group, and a group of carcinomas with other histological types. In conclusion, our results demonstrate the utility of quantitative proteomic analysis for molecular diagnosis and classification of lung cancer cells.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.     

Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.     

Toward a comprehensive quantitative proteome database: protein expression map of lymphoid neoplasms by 2-D DIGE and MS

Using 2-D DIGE, we constructed a quantitative 2-D database including 309 proteins corresponding to 389 protein spots across 42 lymphoid neoplasm cell lines. The proteins separated by 2-D PAGE were identified by MS and assigned to the expression data obtained by 2-D DIGE. The cell lines were categorized into four groups: those from Hodgkin's lymphoma (HL) (4 cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell malignancies (3 cell lines). We characterized the proteins in the database by classifying them according to their expression level. We found 28 proteins with more than a 2-fold difference between the cell line groups. We also noted the proteins that allowed multidimensional separation to be achieved (1) between HL cells and other cells, (2) between the cells derived from B cells, T cells and NK cells, and (3) between HL cells and anaplastic large cell lymphoma cells. Decision tree classification identified five proteins that could be used to classify the 42 cell lines according to differentiation. These results suggest that the quantitative 2-D database using 2-D DIGE will be a useful resource for studying the mechanisms underlying the differentiation phenotypes of lymphoid neoplasms.     

Cloning of GMP-140, a granule membrane protein of platelets and endothelium: sequence similarity to proteins involved in cell adhesion and inflammation

GMP-140 is an integral membrane glycoprotein found in secretory granules of platelets and endothelial cells. After cellular activation, it is rapidly redistributed to the plasma membrane. The cDNA-derived primary structure of GMP-140 predicts a cysteine-rich protein with multiple domains, including a "lectin" region, an "EGF" domain, nine tandem consensus repeats related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Some cDNAs also predict a soluble protein with a deleted transmembrane segment. The domain organization of GMP-140 is similar to that of ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils. This similarity suggests that GMP-140 belongs to a new family of inducible receptors with related structure and function on vascular cells.     

Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade

Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na₂CO₃[pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G₁₁α protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the β-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G_qα/G₁₁α and G_sα, respectively), the distribution and levels of nonstimulated G_i proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150–170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade.     

On the biogenesis of the myelin sheath: Cognate polarized trafficking pathways in oligodendrocytes

Oligodendrocytes, the myelinating cells of the central nervous system, are capable of transporting vast quantities of proteins and of lipids, in particular galactosphingolipids, to the myelin sheath. The sheath is continuous with the plasma membrane of the oligodendrocyte, but the composition of both membrane domains differs substantially. Given its high glycosphingolipid and cholesterol content the myelin sheath bears similarity to the lipid composition of the apical domain of a polarized cell. The question thus arises whether myelin components, like typical apical membrane proteins are transported by an apical-like trafficking mechanism to the sheath, involving a raft-mediated mechanism. Indeed, the evidence indicates the presence of cognate apical and basolateral pathways in oligodendrocytes. However, all major myelin proteins do not participate in this pathway, and remarkably apical-like trafficking seems to be restricted to the oligodendrocyte cell body. In this review, we summarize the evidence on the existence of different trafficking pathways in the oligodendrocyte, and discuss possible mechanisms separating the oligodendrocyte's membrane domains.     

Signaling within a caveolae-like membrane microdomain in human neuroblastoma cells in response to fibroblast growth factor

It is now clear that the plasma membrane is not homogeneous but contains specific subcompartments characterized by their unique lipid and protein composition. Based on their enrichment in various signaling molecules, these microcompartments are now recognized to be sites of localized signal transduction for several extracellular stimuli. At least two different types of microdomains can be identified, largely based on the presence or absence of the caveolin proteins. The generic name of caveolae-like domains is commonly used to refer to both domains indistinguishably. Although caveolin proteins were long thought to be absent from the brain, we have shown that the human neuroblastoma cell line LAN-1 expresses both caveolin-1 and caveolin-2. Basic fibroblast growth factor (FGF)-2 induced a specific signaling response within the caveolae-like domain of LAN-1 cells, characterized by the tyrosine phosphorylation of a 75-80-kDa protein. This protein present in the caveolae-like domains has properties suggesting that it is a member of the SNT family of adapter proteins. The signaling event originating in the caveolae-like domains in response to FGF-2 appeared to require the activation of at least Fyn and Lyn, two members of the Src family of tyrosine kinases. This work suggests that compartmentalized signaling within caveolae-like domains may create a level of specificity for certain growth factors such as FGF.     

Towards functional proteomics of membrane protein complexes: analysis of thylakoid membranes from Chlamydomonas reinhardtii

Functional proteomics of membrane proteins is an important tool for the understanding of protein networks in biological membranes but structural studies on this part of the proteome are limited. In this study we undertook such an approach to analyse photosynthetic thylakoid membranes isolated from wild-type and mutant strains of Chlamydomonas reinhardtii. Thylakoid membrane proteins were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and analysed by immuno-blotting and mass spectrometry for the presence of membrane-spanning proteins. Our data show that light-harvesting complex proteins (LHCP), that cross the membrane with three transmembrane domains, can be separated using this method. We have identified more than 30 different LHCP spots on our gels. Mass spectrometric analysis of 2-DE separated Lhcb1 indicates that this major LHCII protein can associate with the thylakoid membrane with part of its putative transit sequence. Separation of isolated photosystem I (PSI) complexes by 2-DE revealed the presence of 18 LHCI protein spots. The use of two peptide-specific antibodies directed against LHCI subunits supports the interpretation that some of these spots represent products arising from differential processing and post-translational modifications. In addition our data indicate that the reaction centre subunit of PSI, PsaA, that possesses 11 transmembrane domains, can be separated by 2-DE. Comparison between 2-DE maps from thylakoid membrane proteins isolated from a PSI-deficient (Deltaycf4) and a crd1 mutant, which is conditionally reduced in PSI and LHCI under copper-deficiency, showed the presence of most of the LHCI spots in the former but their absence in the latter. Our data demonstrate that (i) hydrophobic membrane proteins like the LHCPs can be faithfully separated by 2-DE, and (ii) that high-resolution 2-DE facilitates the comparative analysis of membrane protein complexes in wild-type and mutants cells.     

Patchwork organization of the yeast plasma membrane into numerous coexisting domains

The plasma membrane is made up of lipids and proteins, and serves as an active interface between the cell and its environment. Many plasma-membrane proteins are laterally segregated in the plane of the membrane, but the underlying mechanisms remain controversial. Here we investigate the distribution and dynamics of a representative set of plasma-membrane-associated proteins in yeast cells. These proteins were distributed non-homogeneously in patterns ranging from distinct patches to nearly continuous networks, and these patterns were in turn strongly influenced by the lipid composition of the plasma membrane. Most proteins segregated into distinct domains. However, proteins with similar or identical transmembrane sequences (TMSs) showed a marked tendency to co-localize. Indeed we could predictably relocate proteins by swapping their TMSs. Finally, we found that the domain association of plasma-membrane proteins has an impact on their function. Our results are consistent with self-organization of biological membranes into a patchwork of coexisting?domains.     

Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.     

A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid

Background

Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way.

Methods

Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization.

Results and Conclusions

We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids.

General significance

This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Amino acid composition of the membrane and aqueous domains of integral membrane proteins

To identify residues which might impart transport capability to the intramembranous regions of transport proteins, we surveyed available data for the 9991 amino acids contained in the aqueous and intramembranous regions of 24 integral membrane proteins: 10 transport (T) proteins and 14 nontransport (NT) proteins. Statistical comparison of percentage occurrence of each amino acid within T and NT samples provided a measure of "typical" composition of T and NT membrane-spanning regions, and showed that the residues partition into membrane and aqueous domains largely in accord with expectation from hydropathy indices. Comparison of aqueous and membrane domain composition between protein categories revealed a statistically similar distribution of residues in aqueous domains, but significant differences in membrane domains: seven residues (Asn, Asp, Gln, Glu, Phe, Pro, Tyr) were preferred in membrane regions of T proteins, and one (Val) was selectively excluded. Chemical and structural considerations suggested that three of these residues--Asn, Tyr, and Pro--are the most likely functional participants in transport processes.     

Eisosome proteins assemble into a membrane scaffold

Spatial organization of membranes into domains of distinct protein and lipid composition is a fundamental feature of biological systems. The plasma membrane is organized in such domains to efficiently orchestrate the many reactions occurring there simultaneously. Despite the almost universal presence of membrane domains, mechanisms of their formation are often unclear. Yeast cells feature prominent plasma membrane domain organization, which is at least partially mediated by eisosomes. Eisosomes are large protein complexes that are primarily composed of many subunits of two Bin-Amphiphysin-Rvs domain-containing proteins, Pil1 and Lsp1. In this paper, we show that these proteins self-assemble into higher-order structures and bind preferentially to phosphoinositide-containing membranes. Using a combination of electron microscopy approaches, we generate structural models of Pil1 and Lsp1 assemblies, which resemble eisosomes in cells. Our data suggest that the mechanism of membrane organization by eisosomes is mediated by self-assembly of its core components into a membrane-bound protein scaffold with lipid-binding specificity.     

Plasma membrane stress induces relocalization of Slm proteins and activation of TORC2 to promote sphingolipid synthesis

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.