Genetic evidence for the temperature-sensing ability of the membrane domain of the Bacillus subtilis histidine kinase DesK

A decrease in environmental temperature leads to the synthesis of Delta5-unsaturated fatty acids in Bacillus subtilis by the fatty acid desaturase Des. Des is regulated by the two-component system DesKR. To understand the mechanism of cold signal perception and transduction by the membrane domain and the cytosolic domain of DesK, we expressed the cytosolic domain of DesK in trans under the control of a xylose-inducible promoter without the membrane domain. We performed growth experiments and a Northern blot analysis. Our results show that the kinase function of the cytosolic domain of DesK is temperature-independent, leading to a constitutive expression of the des gene. These findings support the conclusion that the membrane domain of DesK is the temperature-sensing element of the two-component system.     

Membrane fluidization by alcohols inhibits DesK-DesR signalling in Bacillus subtilis

After cold shock, the Bacillus subtilis desaturase Des introduces double bonds into the fatty acids of existing membrane phospholipids. The synthesis of Des is regulated exclusively by the two-component system DesK/DesR; DesK serves as a sensor of the state of the membrane and triggers Des synthesis after a decrease in membrane fluidity. The aim of our work is to investigate the biophysical changes in the membrane that are able to affect the DesK signalling state. Using linear alcohols (ethanol, propanol, butanol, hexanol, octanol) and benzyl alcohol, we were able to suppress Des synthesis after a temperature downshift. The changes in the biophysical properties of the membrane caused by alcohol addition were followed using membrane fluorescent probes and differential scanning calorimetry.We found that the membrane fluidization induced by alcohols was reflected in an increased hydration at the lipid-water interface. This is associated with a decrease in DesK activity. The addition of alcohol mimics a temperature increase, which can be measured isothermically by fluorescence anisotropy. The effect of alcohols on the membrane periphery is in line with the concept of the mechanism by which two hydrophilic motifs located at opposite ends of the transmembrane region of DesK, which work as a molecular caliper, sense temperature-dependent variations in membrane properties.     

Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast

The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C. and Yaffe, M.P. (1991). J. Cell Biol. 115, 1249-1257). We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.     

Membrane topology of the acyl-lipid desaturase from Bacillus subtilis

The Bacillus subtilis acyl-lipid desaturase (Delta5-Des) is an iron-dependent integral membrane protein, able to selectively introduce double bonds into long chain fatty acids. Structural information on membrane-bound desaturases is still limited, and the present topological information is restricted to hydropathy plots or sequence comparison with the evolutionary related alkane hydroxylase. The topology of Delta5-Des was determined experimentally in Escherichia coli using a set of nine different fusions of N-terminal fragments of Delta5-Des with the reporter alkaline phosphatase (Delta5-Des-PhoA). The alkaline phosphatase activities of cells expressing the Delta5-Des-PhoA fusions, combined with site-directed mutagenesis of His residues identified in most desaturases, suggest that a tripartite motif of His essential for catalysis is located on the cytoplasmic phase of the membrane. These data, together with surface Lys biotinylation experiments, support a model for Delta5-Des as a polytopic membrane protein with six transmembrane- and one membrane-associated domain, which likely represents a substrate-binding motif. This study provides the first experimental evidence for the topology of a plasma membrane fatty acid desaturase. On the basis of our results and the presently available hydrophobicity profile of many acyl-lipid desaturases, we propose that these enzymes contain a new transmembrane domain that might play a critical role in the desaturation of fatty acids esterified in glycerolipids.     

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

Effects of fatty acids on the growth of Caco-2 cells

Epidemiological studies suggest that polyunsaturated fatty acids may protect against colorectal neoplasia. In order to explore this observation, cell proliferation and viability, lipid composition, membrane fluidity, and lipid peroxidation were measured in Caco-2 cells after 48h incubation with various fatty acids. Saturated and monounsaturated fatty acids incorporated less well in the membranes than polyunsaturated fatty acids (PUFAs). All of the PUFAs tested had an inhibitory effect on cell proliferation/viability whereas the saturated and monounsaturated fatty acids did not. Addition of palmitic acid had no significant effect on membrane fluidity whereas unsaturated fatty acids increased membrane fluidity in a dose-dependent manner. PUFAs strongly increased tumor cell lipid peroxidation in a dose-dependent manner. Saturated and monounsaturated fatty acids increased lipid peroxidation in this cell line only at high concentration. Preincubation of Caco-2 cells with vitamin E prevented the inhibition of proliferation/viability, the elevation of the MDA concentration and the increased membrane fluidity induced by PUFAs. Our data indicate that PUFAs are potent inhibitors of the growth of colon cancer cells in vitro.     

Fatty acyl CoA synthetase from Antarctic notothenioid fishes may influence substrate specificity of fat oxidation

Antarctic notothenioid fishes possess large lipid stores that are important fuels for aerobic metabolism. Oxidative muscle tissues of these animals oxidize long-chain mono-unsaturated fatty acids more readily than saturated fatty acids. The mechanistic basis(es) for the substrate specificity of their fatty acid-oxidizing pathway is unknown. We examined the substrate specificity of fatty acyl coenzyme A synthetase (FACS) to determine whether the enzyme contributes to targeting unsaturated fatty acids for preferential transport into mitochondria as fuels for beta-oxidation. Maximal activities of FACS were measured in isolated mitochondria from Notothenia coriiceps and Chaenocephalus aceratus oxidative skeletal muscles in the presence of fatty acids differing in chain lengths and degrees of unsaturation. With the exception of C(22:6), maximal activities were greater with unsaturated substrates than with C(16:0), a saturated fatty acid. Monoenoic fatty acids did not produce the highest activities. Predicted amino acid sequences of FACS from Antarctic C. aceratus, Gobionotothen gibberifrons, and N. coriiceps and sub-Antarctic Notothenia angustata and Eleginops maclovinus were determined to identify amino acid candidates that may be important for determining the substrate specificity of FACS. Substitutions cysteine548 and polar threonine552 within the putative fatty acid binding pocket may contribute to preference for unsaturated fatty acyl substrates compared to saturated fatty acids.     

Effect of membrane fatty acyl composition on LDL metabolism in Hep G2 hepatocytes

The mechanism by which dietary cis-unsaturated fatty acids lower plasma levels of low-density lipoprotein (LDL) cholesterol is unknown. Since plasma membrane incorporation of dietary cis-unsaturated fatty acids is known to alter the function of plasma membrane associated proteins, perhaps by increasing membrane fluidity, we examined LDL receptor function in Hep G2 hepatocytes that were unmodified, enriched with the cis-unsaturated fatty acids oleate or linoleate, or enriched with the saturated fatty acids stearate or palmitate. Hepatocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, uptake, and degradation in comparison to unmodified cells. In contrast, Hep G2 hepatocytes enriched in saturated fatty acids had decreased LDL binding, uptake, and degradation. Enrichment with oleate or linoleate resulted in a decrease in the calculated fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as measured by the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the plasma membrane. Conversely, stearate or palmitate enrichment resulted in an increased plasma membrane fatty acyl mole-weighted melting point and decreased plasma membrane fluidity. LDL binding, uptake, and degradation varied with plasma membrane fluidity in a highly correlated manner. Thus, one mechanism by which dietary cis-unsaturated fatty acids lower LDL cholesterol may possibly involve an alteration in membrane lipid composition or membrane fluidity that promotes enhanced LDL receptor function, thereby leading to increased hepatic clearance of LDL.     

Stress-responsive gene expression in Tetrahymena

Cells properly respond to extracellular stimuli and circumstantial environment. The unicellular eukaryotic protozoan Tetrahymena is a potentially useful animal cell model system for studying the molecular mechanism of adaptation to environment. Tetrahymena is exposed to fluctuations in temperature, pH, amounts of nutrients and concentration of dissolved gases in natural habitat. For example, the cells adapt to cold environment by increase in unsaturated fatty acids in membrane phospholipids to maintain proper membrane fluidity. To accomplish this modification, the activity of fatty acid desaturase is increased upon a down-shift in temperature. We have cloned delta9 fatty acid desaturase which is involved in this process and shown evidence that its mRNA level increased in response to cold environment. Moreover, in order to examine other genes responsive to clod stress, we have adopted mRNA differential display technique to temperature shift-down of T. thermophila. We have cloned two kinase genes, NIMA (never-in-mitosis in Aspergillus nidulans)-related protein kinase (TpNrk) and MAP kinase-related kinase (MRK). Interestingly, these genes were also shown to be expressed by the osmotic stress.