The molecular details of how lipids influence final topological organization of membrane proteins are not well understood. Here, we present evidence that final topology is influenced by lipid–protein interactions most likely outside of the translocon. The N-terminal half of Escherichia coli lactose permease (LacY) is inverted with respect to the C-terminal half and the membrane bilayer when assembled in mutants lacking phosphatidylethanolamine and containing only negatively charged phospholipids. We demonstrate that inversion is dependent on interactions between the net charge of the cytoplasmic surface of the N-terminal bundle and the negative charge density of the membrane bilayer surface. A transmembrane domain, acting as a molecular hinge between the two halves of the protein, must also exit from the membrane for inversion to occur. Phosphatidylethanolamine dampens the translocation potential of negative residues in favor of the cytoplasmic retention potential of positive residues, thus explaining the dominance of positive over negative amino acids as co- or post-translational topological determinants.

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.

The Hippo signaling pathway and its two downstream effectors, the YAP and TAZ transcriptional coactivators, are drivers of tumor growth in experimental models. Studying mouse models, we show that YAP and TAZ can also exert a tumor-suppressive function. We found that normal hepatocytes surrounding liver tumors displayed activation of YAP and TAZ and that deletion of Yap and Taz in these peritumoral hepatocytes accelerated tumor growth. Conversely, experimental hyperactivation of YAP in peritumoral hepatocytes triggered regression of primary liver tumors and melanoma-derived liver metastases. Furthermore, whereas tumor cells growing in wild-type livers required YAP and TAZ for their survival, those surrounded by Yap- and Taz-deficient hepatocytes were not dependent on YAP and TAZ. Tumor cell survival thus depends on the relative activity of YAP and TAZ in tumor cells and their surrounding tissue, suggesting that YAP and TAZ act through a mechanism of cell competition to eliminate tumor cells.

The mechanisms by which lipid bilayer properties govern or influence membrane protein functions are little understood, but a liquid-crystalline state and the presence of anionic and nonbilayer (NB)-prone lipids seem important. An Escherichia coli mutant lacking the major membrane lipid phosphatidylethanolamine (NBprone) requires divalent cations for viability and cell integrity and is impaired in several membrane functions that are corrected by introduction of the "foreign" NBprone neutral glycolipid ␣-monoglucosyldiacylglycerol (MGlcDAG) synthesized by the MGlcDAG synthase from Acholeplasma laidlawii. Dependence on Mg 2؉ was reduced, and cellular yields and division malfunction were greatly improved. The increased passive membrane permeability of the mutant was not abolished, but protein-mediated osmotic stress adaptation to salts and sucrose was recovered by the presence of MGlcDAG. MGlcDAG also restored tryptophan prototrophy and active transport function of lactose permease, both critically dependent on phosphatidylethanolamine. Three mechanisms can explain the observed effects: NB-prone MGlcDAG improves the quenched lateral pressure profile across the bilayer; neutral MGlcDAG dilutes the high anionic lipid surface charge; MGlcDAG provides a neutral lipid that can hydrogen bond and/or partially ionize. The reduced dependence on Mg 2؉ and lack of correction by high monovalent salts strongly support the essential nature of the NB properties of MGlcDAG.The lipid bilayer of biological membranes acts as a permeability barrier permitting maintenance of essential ion gradients and is also the local environment for integral and peripheral membrane proteins. Important bilayer structural features are a liquid-crystalline state, an optimal length of the lipid chains, and critical fractions of anionic and nonbilayer-(NB) 1 prone lipids. Because of their small head groups the NB-prone lipids, when embedded in a bilayer, cause a curvature elastic stress of each monolayer with closer acyl chain packing (i.e. higher chain order) that changes the lateral pressure profile across the membrane (1). Increasing the proportion of NBprone lipids leads to a lower temperature for the bilayer to NB phase transition, which in many cases is fairly close to the growth temperature of organisms (2, 3). The balance of bilayer to NB-prone lipids may affect the passive membrane permeability of the bilayer (e.g. (4)), the folding and assembly of membrane proteins (5), and the function of important cellular processes such as cell division, transport, and osmotic responses. The essential character of such NB-prone lipids for cell membrane functions has been little studied.NB-prone lipids substantially affect the activity of several different types of proteins when studied in vitro. For example, a critical conformational change of transmembrane rhodopsin (6) and the activity of the interface-bound CTP:phosphocholine cytidylyltransferase (7) are modulated by variations in NBprone lipids and membrane curvature elastic stress, respectively. Likewise...