Yeast phosphatidic acid phosphatase Pah1 hops and scoots along the membrane phospholipid bilayer

PA phosphatase, encoded by PAH1 in the yeast Saccharomyces cerevisiae, catalyzes the Mg2+-dependent dephosphorylation of PA, producing DAG at the nuclear/ER membrane. This enzyme plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. As an interfacial enzyme, PA phosphatase interacts with the membrane surface, binds its substrate, and catalyzes its reaction. The Triton X-100/PA-mixed micellar system has been utilized to examine the activity and regulation of yeast PA phosphatase. This system, however, does not resemble the in vivo environment of the membrane phospholipid bilayer. We developed an assay system that mimics the nuclear/ER membrane to assess PA phosphatase activity. PA was incorporated into unilamellar phospholipid vesicles (liposomes) composed of the major nuclear/ER membrane phospholipids, PC, PE, PI, and PS. We optimized this system to support enzyme-liposome interactions and to afford activity that is greater than that obtained with the aforementioned detergent system. Activity was regulated by phospholipid composition, whereas the enzyme’s interaction with liposomes was insensitive to composition. Greater activity was attained with large (≥100 nm) versus small (50 nm) vesicles. The fatty-acyl moiety of PA had no effect on this activity. PA phosphatase activity was dependent on the bulk (hopping mode) and surface (scooting mode) concentrations of PA, suggesting a mechanism by which the enzyme operates along the nuclear/ER membrane in vivo.

Identification of a Δ11 desaturase from the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal fungi are oleaginous organisms and the most abundant fatty acyl moiety usually found in their lipids is palmitvaccenic acid (16:1Δ11cis). However, it is not known how this uncommon fatty acid species is made. Here we have cloned two homologs of Lepidopteran fatty acyl-CoenzymeA Δ11 desaturases from Rhizophagus irregularis. Both DES1 and DES2 are expressed in intraradicle mycelium and can complement the unsaturated fatty acid-requiring auxotrophic growth phenotype of the Saccharomyces cerevisiae ole1Δ mutant. DES1 expression leads almost exclusively to oleic acid (18:1Δ9cis) production, whereas DES2 expression results in the production of 16:1Δ11cis and vaccenic acid (18:1Δ11cis). DES2 therefore encodes a Δ11 desaturase that is likely to be responsible for the synthesis of 16:1Δ11cis in R. irregularis.

Aging results in paradoxical susceptibility of fat cell progenitors to lipotoxicity

Aging is associated with metabolic syndrome, tissue damage by cytotoxic lipids, and altered fatty acid handling. Fat tissue dysfunction may contribute to these processes. This could result, in part, from age-related changes in preadipocytes, since they give rise to new fat cells throughout life. To test this hypothesis, preadipocytes cultured from rats of different ages were exposed to oleic acid, the most abundant fatty acyl moiety in fat tissue and the diet. At fatty acid concentrations at which preadipocytes from young animals remained viable, cells from old animals accumulated lipid in multiple small lipid droplets and died, with increased apoptotic index, caspase activity, BAX, and p53. Rather than inducing apoptosis, oleic acid promoted adipogenesis in preadipocytes from young animals, with appearance of large lipid droplets. CCAAT/enhancer-binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ) increased to a greater extent in cells from young than old animals after oleate exposure. Oleic acid, but not glucose, oxidation was impaired in preadipocytes and fat cells from old animals. Expression of carnitine palmitoyltransferase (CPT)-1, which catalyzes the rate-limiting step in fatty acid β-oxidation, was not reduced in preadipocytes from old animals. At lower fatty acid levels, constitutively active CPT I expression enhanced β-oxidation. At higher levels, CPT I was not as effective in enhancing β-oxidation in preadipocytes from old as young animals, suggesting that mitochondrial dysfunction may contribute. Consistent with this, medium-chain acyl-CoA dehydrogenase expression was reduced in preadipocytes from old animals. Thus preadipocyte fatty acid handling changes with aging, with increased susceptibly to lipotoxicity and impaired fatty acid-induced adipogenesis and β-oxidation.

Phospholipid composition of several clinically relevant Corynebacterium species as determined by mass spectrometry: an unusual fatty acyl moiety is present in inositol-containing phospholipids of Corynebacterium urealyticum

A comparative study on phospholipids of Corynebacterium amycolatum, Corynebacterium jeikeium and Corynebacterium urealyticum was carried out using fast-atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry. Data obtained indicate the presence of acylphosphatidylglycerol (APG), diphosphatidylglycerol, phosphatidylglycerol (PG), phosphatidylinositol (PI) and triacylphosphatidylinositol dimannosides (Ac3PIM2) in these bacteria. In general, octadecenoyl and hexadecanoyl fatty acyl moieties predominated in phospholipids of C. amycolatum, whereas high levels of hexadecenoyl were found in C. jeikeium and C. urealyticum. Mass spectra from purified APG and PG indicated that the sn-1 position of the glycerol was occupied by octadecenoyl in the three species studied. Notably, several major molecular species of PI and Ac3PIM2 from C. urealyticum contained significant amounts of a moiety identified as 10-methyleneoctadecanoyl, located at the sn-1 position of these molecules. On the other hand, multiantibiotic resistant and susceptible strains of C. amycolatum differed in several minor phospholipid fatty acids of 19 carbon atoms, identified as 10-methyloctadecenoic, 10-methyloctadecanoic (tuberculostearic acid) and 10-methyleneoctadecanoic. The results demonstrate an overall similarity among the phospholipids of the different species studied but also significant differences related to the acyl chains of the glycerol moiety of these compounds, notably the high levels of an unusual fatty acyl moiety in inositol-containing phospholipids of C. urealyticum.

Chitin oligosaccharides can induce cortical cell division in roots of Vicia sativa when delivered by ballistic microtargeting

Rhizobia, bacterial symbionts of leguminous plants, produce lipo-chitin oligosaccharide (LCO) signal molecules that can induce nodule organogenesis in the cortex of legume roots in a host-specific way. The multi-unsaturated fatty acyl and the O-acetyl moieties of the LCOs of Rhizobium leguminosarum biovar viciae were shown to be essential for obtaining root nodule induction in Vicia sativa plants. We have used ballistic microtargeting as a novel approach to deliver derivatives of the nodulation signal molecules inside the roots of V. sativa. This method offers the unique ability to introduce soluble compounds into the tissue at a small area. The mitogenic effect of microtargeting of chitin oligosaccharides, including an analysis of the influence of the chain length and modifications, was tested in a qualitative assay. The role of a cell division factor from the root stele, uridine, has also been examined in these experiments. The results show that O-acetylated chitin oligosaccharides can induce root cortical cell divisions when delivered by microtargeting. For this effect it is essential that uridine is co-targeted. The foci of cortical cell division were often similar to root nodule primordia. Anatomical examination also revealed chimeric structures that share characteristics with lateral root and nodule primordia. Our data favour a model in which the oligosaccharide moiety of the rhizobial LCO induces cortical cell division and the fatty acyl moiety plays a role in transport of the LCO into the plant tissue.

Microstructural morphology of sphingolipid dispersions as investigated by freeze-fracture EM

The biological significance of the sphingomyelins (SM) and monoglycosylated sphingolipids like galactosylceramides (GalCer) are well documented Our recent investigation showed tubular bilayers in the aqueous dispersions of N-nervonoyl GalCer [N-(24:lΔ15,cls) GalCer] (a major fatty acyl moiety of natural GalCer). To determine the influence of lipid head groups on the resulting mesophasic morphology, we investigated microstructural self-assemblies of N-nervonoyl-SM [N-(24:1 Δ15,cls) SM; the second most abundant sphingomyelin in mammalian cell membranes], 1- palmitoyl-2-nervonoyl phosphatidylcholine [PNPC] (the lipid species with the same acyl chain configuration as in N-(24: 1) GalCer) and also compared it with egg-SM by freeze-fracture EM.Procedures for synthesizing and purifying N-(24:1) GalCer, N-(24:1) SM, and PNPC have been reported . Egg-SM was purchased from Avanti Polar Lipids, Alabaster AL. All lipids were >99% pure as checked by thin layer chromatography. Lipid dispersions were prepared by hydrating dry lipid with phosphate buffer (pH 6.6) at 80-90°C (3-5 min), vigorously vortexing (1 min) and repeating this procedure for three times prior to three freeze-thaw cycles.

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.

Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function.

Two proteins in the yeast Saccharomyces cerevisiae that are encoded by the genes RAS1 and RAS2 are structurally and functionally homologous to proteins of the mammalian ras oncogene family. We examined the role of fatty acylation in the maturation of yeast RAS2 protein by creating mutants in the putative palmitate addition site located at the carboxyl terminus of the protein. Two mutations, Cys-318 to an opal termination codon and Cys-319 to Ser-319, were created in vitro and substituted in the chromosome in place of the normal RAS2 allele. These changes resulted in a failure of RAS2 protein to be acylated with palmitate and a failure of RAS2 protein to be localized to a membrane fraction. The mutations yielded a Ras2- phenotype with respect to the ability of the resultant mutants to grow on nonfermentable carbon sources and to complement ras1- mutants. However, overexpression of the ras2Ser-319 product yielded a Ras+ phenotype without a corresponding association of the mutant protein with the membrane fraction. We conclude that the presence of a fatty acyl moiety is important for localizing RAS2 protein to the membrane where it is active but that the fatty acyl group is not an absolute requirement of RAS2 protein function.