Pablo S. Aguilar

Molecular basis of thermosensing: a two‐component signal transduction thermometer in Bacillus subtilis

Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock‐induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two‐component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Δ5‐lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position −28 to −77 relative to the start site of the temperature‐regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Δ5‐desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK–DesR two‐component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

Eisosomes mark static sites of endocytosis

Endocytosis functions to recycle plasma membrane components, to regulate cell-surface expression of signalling receptors and to internalize nutrients in all eukaryotic cells. Internalization of proteins, lipids and other cargo can occur by one of several pathways that have different, but often overlapping, molecular requirements. To mediate endocytosis, effectors assemble transiently underneath the plasma membrane, carry out the mechanics of membrane deformation, cargo selection and vesicle internalization, and then disassemble. The mechanism by which endocytosis initiates at particular locations on the plasma membrane has remained unknown. Sites of endocytosis might be formed randomly, induced by stochastic protein and/or lipid clustering. Alternatively, endocytosis might initiate at specific locations. Here we describe large immobile protein assemblies at the plasma membrane in the yeast Saccharomyces cerevisiae that mark endocytic sites. These structures, termed eisosomes (from the Greek ‘eis’, meaning into or portal, and ‘soma’, meaning body), are composed primarily of two cytoplasmic proteins, Pil1 and Lsp1. A plasma membrane protein, Sur7, localizes to eisosomes. These structures colocalize with sites of protein and lipid endocytosis, and their components genetically interact with known endocytic effectors. Loss of Pil1 leads to clustering of eisosome remnants and redirects endocytosis and endocytic effector proteins to these clusters.

Control of fatty acid desaturation: a mechanism conserved from bacteria to humans

Unsaturated fatty acids (UFAs) have profound effects on the fluidity and function of biological membranes. Microorganisms, plants and animals regulate the synthesis of UFAs during changing environmental conditions as well as in response to nutrients. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturase gene transcription through signalling pathways that are governed by sensors embedded in cellular membranes. Here, we review recently discovered components of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeasts and animals that indicate an ancient role of transmembrane signalling mechanisms and integrate membrane composition with lipid biosynthesis.

Genetic basis of cell–cell fusion mechanisms

Cell-cell fusion in sexually reproducing organisms is a mechanism to merge gamete genomes and, in multicellular organisms, it is a strategy to sculpt organs, such as muscle, bone, and placenta. Moreover, this mechanism has been implicated in pathological conditions, such as infection and cancer. Studies of genetic model organisms have uncovered a unifying principle: cell fusion is a genetically programmed process. This process can be divided in three stages: competence (cell induction and differentiation); commitment (cell determination, migration, and adhesion); and cell fusion (membrane merging and cytoplasmic mixing). Recent work has led to the discovery of fusogens, which are cell fusion proteins that are necessary and sufficient to fuse cell membranes. Two unrelated families of fusogens have been discovered, one in mouse placenta and one in Caenorhabditis elegans (syncytins and F proteins, respectively). Current research aims to identify new fusogens and determine the mechanisms by which they merge membranes.

Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl‐lipid desaturase

Summary The Des pathway of Bacillus subtilis regulates the expression of the acyl‐lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids (UFAs) from saturated phospholipid precursors. Previously, we showed that the master switch for the Des pathway is a two‐component regulatory system composed of a membrane‐associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. Activation of this pathway takes place when cells are shifted to low growth temperature. Here, we report on the mechanism by which isoleucine regulates the Des pathway. We found that exogenous isoleucine sources, as well as its α‐keto acid derivative, which is a branched‐chain fatty acid precursor, negatively regulate the expression of the des gene at 37°C. The DesK–DesR two‐component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37°C. Fatty acid profiles strongly indicate that isoleucine affects the signalling state of the DesK sensor protein by dramatically increasing the incorporation of the lower‐melting‐point anteiso‐branched‐chain fatty acids into membrane phospholipids. We propose that both a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism. Thus, the Des pathway would provide a novel mechanism to optimize membrane lipid fluidity at a constant temperature.

A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling

The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function.

Pkh-kinases control eisosome assembly and organization

Eisosomes help sequester a subgroup of plasma membrane proteins into discrete membrane domains that colocalize with sites of endocytosis. Here we show that the major eisosome component Pil1 in vivo is a target of the long‐chain base (LCB, the biosynthetic precursors to sphingolipids)‐signaling pathway mediated by the Pkh‐kinases. Eisosomes disassemble if Pil1 is hyperphosphorylated (i) upon overexpression of Pkh‐kinases, (ii) upon reducing LCB concentrations by inhibiting serine‐palmitoyl transferase in lcb1‐mutant cells or by poisoning the enzyme with myriocin, and (iii) upon mimicking hyperphosphorylation in pil1‐mutant cells. Conversely, more Pil1 assembles into eisosomes if Pil1 is hypophosphorylated (i) upon reducing Pkh‐kinase activity in pkh1 pkh2‐mutant cells, (ii) upon activating Pkh‐kinases by addition of LCBs, and (iii) upon mimicking hypophosphorylation in pil1‐mutant cells. The resulting enlarged eisosomes show altered organization. Other data suggest that Pkh signaling and sphingolipids are important for endocytosis. Taken together with our previous results that link eisosomes to endocytosis, these observations suggest that Pkh‐kinase signaling relayed to Pil1 may help regulate endocytic events to modulate the organization of the plasma membrane.

A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of ∼400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.

The eisosome core is composed of BAR domain proteins

The core components of eisosomes, Pil1 and Lsp1, are membrane-sculpting BAR proteins. In addition, TORC2 substrates Slm1 and Slm2 have F-BAR domains that are needed for targeting into eisosomes. Results support a model in which BAR domain protein-mediated membrane bending leads to domain formation within the plasma membrane.

The Bacillus subtilis Acyl Lipid Desaturase Is a Δ5 Desaturase

Bacillus subtilis was recently reported to synthesize unsaturated fatty acids (UFAs) with a double bond at positions Δ5, Δ7, and Δ9 (M. H. Weber, W. Klein, L. Muller, U. M. Niess, and M. A. Marahiel, Mol. Microbiol. 39:1321-1329, 2001). Since this finding would have considerable importance in the double-bond positional specificity displayed by the B. subtilis acyl lipid desaturase, we have attempted to confirm this observation. We report that the double bond of UFAs synthesized by B. subtilis is located exclusively at the Δ5 position, regardless of the growth temperature and the length chain of the fatty acids.