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Biomedical subjects

G B Wells

Publications and source records attributed to G B Wells.

At least 19 recordsLinked to original sources
PubMed2000

Assembly and ligand binding properties of the water-soluble extracellular domains of the glutamate receptor 1 subunit.

High resolution structural studies of models of glutamate receptors (GluRs) have been limited to monomeric models of the ligand-binding site. To obtain oligomeric models of glutamate receptors that can reveal more complete structural information, we examined the assembly and ligand binding properties of two truncated versions of the GluR1 subunit. The first version, GluR1-WS, consisted of only the N-terminal extracellular segment (Ala(1)-Glu(520)) bridged by a synthetic linker to the second extracellular domain (Asn(615)-Gly(790)). The second version, GluR1-M1, consisted of the first N-terminal extracellular domain (Ala(1)-Glu(520)) bridged by a synthetic linker to a second segment containing the second extracellular domain, the third transmembrane domain, and the intracellular C-terminal domain (Asn(615)-Leu(889)). When expressed in Xenopus oocytes, GluR-WS was secreted and water-soluble; GluR1-M1 was displayed on the surface of oocytes. GluR1-WS exhibited a velocity sedimentation profile that was consistent with assembly of homooligomers and bound the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid with high affinity. These findings show that the extracellular domains of GluR1 that are sufficient for ligand binding apparently are sufficient for subunit assembly and might be a suitable target for structural studies of a water-soluble GluR1 oligomer.

Animals↗
PubMed1999

The effects of ibogaine on dopamine and serotonin transport in rat brain synaptosomes.

Ibogaine has been shown to affect biogenic amine levels in selected brain regions. Because of the involvement of these neurotransmitters in drug addiction, the effects of ibogaine on biogenic amine transport may contribute to the potential anti-addictive properties of ibogaine in vivo. With rat brain synaptosomes as our experimental system, we measured the effects of ibogaine on the uptake and release of dopamine (DA) and serotonin (5-HT). Ibogaine competitively blocked both DA and 5-HT uptake with IC50 values of 20 microM at 75 nM 3H-DA and 2.6 microM at 10 nM 3H-5-HT. Ibogaine had no effect on K+-induced release of 3H-DA from preloaded synaptosomes, but 20 microM and 50 microM ibogaine inhibited roughly 40% and 60%, respectively, of the K(+)-induced release of 3H-5-HT from preloaded synaptosomes. In the absence of a depolarizing stimulus, ibogaine evoked a small release of 3H-DA but not 3H-5-HT. These relatively low-potency effects of ibogaine on DA and 5-HT uptake in synaptosomes are consistent with the low binding affinity of ibogaine that has been previously reported for DA and 5-HT transporters. Our results show that if ibogaine modulates DA and 5-HT levels in the brain by directly blocking their uptake, then a concentration of ibogaine in the micromolar range is required. Furthermore, if the anti-addictive effects of ibogaine require this concentration, then ibogaine likely exerts these effects through a combination of neurotransmitter pathways, because binding affinities and functional potencies of ibogaine in the micromolar range have been reported for a variety of neuronal receptors and transporters.

Animals↗
PubMed1998

Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis.

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.

Antifungal Agents↗
PubMed1998

Water-soluble nicotinic acetylcholine receptor formed by alpha7 subunit extracellular domains.

Water-soluble models of ligand-gated ion channels would be advantageous for structural studies. We investigated the suitability of three versions of the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor (AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first transmembrane domain and was soluble without detergent. For all three, their equilibrium binding affinities for alpha-bungarotoxin, nicotine, and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the alpha7 ECD can form a water-soluble AChR that is a model of the ECD of the full-length alpha7 AChR.

Acetylcholine↗
PubMed1998

Response of CD4+ T cells from myasthenic patients and healthy subjects of biosynthetic and synthetic sequences of the nicotinic acetylcholine receptor.

We investigated the suitability of pools of overlapping synthetic peptides spanning the complete alpha 1 subunit sequence of the human muscle acetylcholine receptor (AChR) (alpha 1 pool) or the extracellular domain (residues 1-218, alpha 11-218 pool), and of biosynthetic alpha 1 constructs from E. coli, as stimulants of human CD4+ cells from myasthenia gravis (MG) patients and healthy subjects. A construct corresponding to residues alpha 11-209 was obtained as solubilized inclusion bodies (ib alpha 11-209), or purified by SDS gel electrophoresis (pur alpha 11-209). A second construct included the extracellular, cytoplasmic and carboxylterminal domains plus histidine residues, and was obtained as inclusion bodies (ib alpha 1NoTrans) or purified by gel permeation and histidine tag affinity chromatography (pur alpha 1NoTrans). A biosynthetic extracellular domain of the neuronal AChR alpha 7 subunit (ib alpha 71-206) isolated from E. coli as inclusion bodies served as control for bacterial contaminants. We used ib alpha 11-209, pur alpha 11-209 and peptide pools to propagate CD4+ lines from two MG patients. The lines obtained using pur alpha 11-209 and the peptide pools recognized the peptide pools and alpha 1 constructs tested well, but ib alpha 71-206 poorly or not at all. These lines recognized peptides known to form CD4+ epitopes in these patients. The ib alpha 11-209 lines recognized ib alpha 11-209 and ib alpha 71-206 strongly, but recognized poorly pur alpha 11-209 and the alpha 11-218 pool. We propagated T-cell lines from a healthy subject using pur alpha 11-209 and ib alpha 11-209. The pur alpha 11-209 line recognized pur alpha 11-209 and the alpha 11-218 pool, but not ib alpha 11-209 or ib alpha 71-206. The ib alpha 11-209 line recognized ib alpha 11-209 and ib alpha 71-206, but not pur alpha 11-209 or the alpha 11-218 pool. We tested blood CD4+ cells from six MG patients and eight healthy subjects with ib alpha 11-209, pur alpha 11-209, the alpha 11-218 pool and--in the healthy subjects--also ib alpha 71-206, ib alpha 1NoTrans and pur alpha 1NoTrans. In both populations, the alpha 11-218 pool elicited low and sporadic responses, while the constructs elicited clear responses that were frequently higher for ib alpha 11-209 than pur alpha 11-209. The responses to ib alpha 71-206 were strong and comparable to those to ib alpha 11-209, ib alpha 1NoTrans, and pur alpha 1NoTrans. These results indicate that even purified constructs from E. coli contain bacterial contaminants recognized by CD4+ cells. They should not be used to test unselected blood CD4+ cells, because they may evoke strong CD4+ responses to the bacterial antigens. Purified recombinant sequences may be suitable for propagation of CD4+ cell lines, if the specificity of the lines can be verified using different antigen preparations. Short synthetic peptide sequences can be safely used for propagation of specific CD4+ cells. Although they are poor stimulants for unselected blood CD4+ cells, the low responses they elicit are probably due to these cells.

Amino Acid Sequence↗
PubMed1998

Suprasellar osteolipoma: case report.

BACKGROUND: Osteolipomas are distinguished from other intracranial lipomas by their arrangement of central adipose and peripheral osseous tissues and by characteristically arising in the suprasellar/interpeduncular region. METHODS: We report computed tomography (CT), magnetic resonance imaging (MRI), and pathology findings from this 34-year-old man who underwent surgical removal of this benign lesion. RESULTS: This case displays the distinctive histopathology that has been reported in 13 of 31 (42%) lipomas in this region. In contrast, ossification of lipomas at other intracranial sites is relatively rare. CONCLUSIONS: Ossification should be expected in many suprasellar/interpeduncular lipomas, and osteolipoma should be included in the radiologic differential diagnosis of fat-intensity masses with calcification in this region.

Adult↗
PubMed1998

Determinants of channel gating located in the N-terminal extracellular domain of nicotinic alpha7 receptor.

We identified regions within the N-terminal extracellular domain of alpha7 nicotinic acetylcholine receptors that affect channel gating. By single-channel analysis of alpha7 nicotinic acetylcholine receptors currents, we show that the difference in efficacy between the two agonists acetylcholine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) is due to a slower channel activation rate by DMPP. The partial efficacy of DMPP was not caused by channel block or faster desensitization of alpha7 AChRs by DMPP. In addition, the efficacy and, by inference, the activation rate were found to be voltage dependent. Using chimeras of the two closely related subunits alpha7 and alpha8, we map residues that affect channel activation rate and agonist affinity to two different regions of the extracellular domain. Residues that affect channel activation rate are within the sequence 1-179, whereas residues that affect agonist affinity are within the sequence 180-208.

Animals↗
PubMed1997

Sphingolipids are potential heat stress signals in Saccharomyces.

The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways. The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses. Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiae heat stress response. Our data show a 2-3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress. Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate. Dihydrosphingosine induces expression of a STRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals. The TPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness. Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S. cerevisiae and quantify the importance of each during heat stress.

Depsipeptides↗
PubMed1997

Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene.

Knowledge of the Saccharomyces cerevisiae genes and proteins necessary for sphingolipid biosynthesis is far from complete. Such information should expedite studies of pathway regulation and sphingolipid functions. Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frame YDR072c), termed Ipt1 (inositolphosphotransferase 1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S. cerevisiae. This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide. The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate [3H-inositol]phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase). M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity. An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions. Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases.

Amino Acid Sequence↗
PubMed1997

Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene.

We have identified a Saccharomyces cerevisiae gene necessary for the step in sphingolipid synthesis in which inositol phosphate is added to ceramide to form inositol-P-ceramide, a reaction catalyzed by phosphatidylinositol:ceramide phosphoinositol transferase (IPC synthase). This step should be an effective target for antifungal drugs. A key element in our experiments was the development of a procedure for isolating mutants defective in steps in sphingolipid synthesis downstream from the first step including a mutant defective in IPC synthase. An IPC synthase defect is supported by data showing a failure of the mutant strain to incorporate radioactive inositol or N-acetylsphinganine into sphingolipids and, by using an improved assay, a demonstration that the mutant strain lacks enzyme activity. Furthermore, the mutant accumulates ceramide when fed exogenous phytosphingosine as expected for a strain lacking IPC synthase activity. Ceramide accumulation is accompanied by cell death, suggesting the presence of a ceramide-activated death response in yeast. A gene, AUR1 (YKL004w), that complements the IPC synthase defect and restores enzyme activity and sphingolipid synthesis was isolated. Mutations in AUR1 had been shown previously to give resistance to the antifungal drug aureobasidin A, leading us to predict that the drug should inhibit IPC synthase activity. Our data show that the drug is a potent inhibitor of IPC synthase with an IC50 of about 0.2 nM. Fungal pathogens are an increasing threat to human health. Now that IPC synthase has been shown to be the target for aureobasidin A, it should be possible to develop high throughput screens to identify new inhibitors of IPC synthase to combat fungal diseases.

Amino Acid Sequence↗
PubMed1997

Ion selectivity predictions from a two-site permeation model for the cyclic nucleotide-gated channel of retinal rod cells.

We developed a two-site, Eyring rate theory model of ionic permeation for cyclic nucleotide-gated channels (CNGCs). The parameters of the model were optimized by simultaneously fitting current-voltage (IV) data sets from excised photoreceptor patches in electrolyte solutions containing one or more of the following ions: Na+, Ca2+, Mg2+, and K+. The model accounted well for 1) the shape of the IV relations; 2) the binding affinity for Na+; 3) reversal potential values with single-sided additions of Ca2+ or Mg2+ and biionic KCl; and 4) the K1 and voltage dependence for divalent block from the cytoplasmic side of the channel. The differences between the predicted K1's for extracellular block by Ca2+ and Mg2+ and the values obtained from heterologous expression of only the alpha-subunit of the channel suggest that the beta-subunit or a cell-specific factor affects the interaction of divalent cations at the external but not the internal face of the channel. The model predicts concentration-dependent permeability ratios with single-sided addition of Ca2+ and Mg2+ and anomalous mole fraction effects under a limited set of conditions for both monovalent and divalent cations. Ca2+ and Mg2+ are predicted to carry 21% and 10%, respectively, of the total current in the retinal rod cell at -60 mV.

Ambystoma↗
PubMed1996

Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits.

Nicotinic acetylcholine receptors formed from combinations of alpha3, beta2, beta4, and alpha5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human alpha3, beta2, beta4, and alpha5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of alpha3beta2, alpha3beta4, alpha3beta2alpha5, and alpha3beta4alpha5 subunits but not for other combinations of one, two, or three of these subunits. alpha5 subunits assembled inside the oocyte with beta2 but not with alpha3 subunits or other alpha5 subunits. alpha5 subunits coassembled very efficiently with alpha3beta2 or alpha3beta4 combinations. The presence of alpha5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. The presence of alpha5 subunits increased the rate of desensitization of both receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. In the case of receptors containing alpha3 and beta4 subunits, the addition of alpha5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing alpha3 and beta2 subunits, the addition of alpha5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 microM and the EC50 for nicotine from 6.8 to 1.9 microM, while increasing the efficacy of nicotine from 50% on alpha3beta2 receptors to 100% on alpha3beta2alpha5 receptors. Both alpha3beta2 and alpha3beta2alpha5 receptors expressed in oocytes sedimented at the same 11 S value as native alpha3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma alpha3, beta2, and alpha5 subunits were co-assembled, and 56% of the receptor subtypes containing alpha3 subunits also contained beta2 subunits. The beta2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from alpha3 and beta2 or alpha3, beta2, and alpha5 subunits rather than the low affinity exhibited by receptors formed from alpha3 and beta4 or alpha3, beta4, and alpha5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing alpha3 subunits in SH-SY5Y cells. The affinities of alpha3beta2 receptors expressed in oocytes were similar to the affinities of native alpha3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Acetylcholine↗
PubMed1996

Isolation and composition of inositolphosphorylceramide-type sphingolipids of hyphal forms of Candida albicans.

Hyphal forms of the human pathogen Candida albicans have been found to contain substantial quantities of phosphosphingolipids. These lipids were fractionated into three classes by normal-phase high-performance liquid chromatography. The first class contained equimolar amounts of phosphorus, inositol, phytosphingosines, and fatty acids; their composition and chromatographic behavior suggest that these compounds are inositolphosphorylceramides. The second class contained equimolar amounts of phosphorus, mannosylinositol, phytosphingosines, and fatty acids; their composition and chromatographic behavior indicate that these compounds are mannosylinositolphosphorylceramides. The third class of compounds contained phosphorus, mannosylinositol, inositol, phytosphingosines, and fatty acids in a molar ratio of 2:1:1:1:1; their composition and chromatographic behavior indicate that these compounds are mannosyldiinositolphosphorylceramides. Molecular species in each class differ in the composition of long chain bases and fatty acids; the most abundant long chain bases were C18 and C20 phytosphingosines, and the most abundant fatty acids were hydroxy and nonhydroxy C24-26. The array of sphingolipids in C. albicans is similar to that of Saccharomyces cerevisiae. Sphingolipids have been shown to be essential in S. cerevisiae, thus these lipids, which are not present in animals, offer a potentially unique target for antifungal chemotherapy against C. albicans.

Alkalies↗
PubMed1994

The LCB2 gene of Saccharomyces and the related LCB1 gene encode subunits of serine palmitoyltransferase, the initial enzyme in sphingolipid synthesis.

The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2- to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms.

Acyltransferases↗
PubMed1994

Structure at the active site of an acylenzyme of alpha-chymotrypsin and implications for the catalytic mechanism. An electron nuclear double resonance study.

The structure of the acylenzyme intermediate in the hydrolysis of the specific spin-label ester substrate methyl N-(2,2,5,5-tetramethyl-1-oxypyrrolinyl-3-carbonyl)-L-tryptophan ate and its fluoro analogs catalyzed by alpha-chymotrypsin (EC 3.4.21.1) has been determined by electron nuclear double resonance (ENDOR) and molecular modeling methods. By a combination of kinetic and cryoenzymological methods, we have established conditions to stabilize the spin-labeled acylenzyme reaction intermediate. Proton ENDOR features specific for the substrate were assigned on the basis of specific deuteration. From the observed ENDOR shifts for protons and fluorines that correspond to principal hyperfine coupling components, the dipolar hyperfine coupling contributions were calculated to estimate electron-nucleus distances. With these dipolar separations as constraints, conformations of the substrate both free in solution and in the active site of alpha-chymotrypsin were determined by molecular graphics analysis. Comparison of the conformation of the bound substrate to that of the free substrate showed that formation of the acylenzyme requires significant torsional alteration in substrate structure. The structural relationships between active-site residues and the substrate in its ENDOR-assigned conformation are examined with respect to the requirements of stereoelectronic rules for formation and breakdown of the acylenzyme species.

Acylation↗
PubMed1994

Lhermitte-Duclos disease and Cowden's syndrome in an adolescent patient. Case report.

Recent reports of seven cases of Lhermitte-Duclos disease occurring in adult patients with Cowden's syndrome (multiple hamartoma syndrome) strongly suggest that Lhermitte-Duclos disease is one of the types of neoplasia that characterize this syndrome. A case of Lhermitte-Duclos disease is reported in a 16-year-old girl with craniomegaly, choroidal hamartoma, and conjunctival papilloma of the right eye, and a history of bilateral multinodular adenomatous goiter and cystic hygroma. These findings strongly suggest a diagnosis of Cowden's syndrome. Although the syndrome traditionally has been defined by mucocutaneous criteria, it typically also involves hamartomas and neoplasia of internal organs, most commonly in the thyroid, breast, and female genitourinary tract. Because the mucocutaneous features may develop several decades after birth, the present case both supports the previously reported association between Lhermitte-Duclos disease and Cowden's syndrome and highlights the need for long-term follow-up monitoring of a pediatric patient with Lhermitte-Duclos disease because of the risk of malignancies associated with Cowden's syndrome.

Adolescent↗
PubMed1993

A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase.

Saccharomyces cerevisiae normally requires sphingolipid biosynthesis for growth; however, mutant strains lacking sphingolipids have been isolated by suppression of a genetic defect in sphingolipid long chain base biosynthesis. To begin to understand the nature of the suppressor(s) we isolated and characterized a suppressor gene, SLC1 (sphingolipid compensation). DNA sequence analysis showed that the wild type SLC1 allele differs from the suppressor allele by a single nucleotide which changes Gln-44 in the predicted wild type protein to Leu4-4 in the predicted SLC1-1 suppressor protein. The predicted SLC1 protein sequence is homologous to the 1-acyl-sn-glycerol-3-phosphate acyltransferase of Escherichia coli encoded by the plsC gene. The homology extends to function as well since the SLC1 gene complements the growth defect in an E. coli strain mutated in plsC. These results suggest that the SLC1 protein has a fatty acyltransferase activity. SLC1 thus may be the first eucaryotic sn2-acylglyceride fatty acyltransferase gene to be cloned. SLC strains grown in the absence of long chain base make novel phosphatidylinositol derivatives (Lester, R. L., Wells, G. B., Oxford, G., and Dickson, R. C. (1993) J. Biol. Chem. 268, 845-856) having a C26 fatty acid at the sn-2 position and the same polar head groups as normal sphingolipids. We postulate that the SLC1 suppressor allele encodes a variant enzyme with an altered substrate specificity that enables it to use a C26 in place of a C16/18 fatty acid precursor to acylate the sn-2 position of inositol-containing glycerolipids.

Acyltransferases↗
PubMed1993

Mutant strains of Saccharomyces cerevisiae lacking sphingolipids synthesize novel inositol glycerophospholipids that mimic sphingolipid structures.

Mutant strains of Saccharomyces cerevisiae, termed SLC, make no detectable sphingolipids when cultured without a sphingolipid long chain base such as phytosphingosine (Dickson, R.C., Wells, G.B., Schmidt, A., and Lester, R.L. (1990) Mol. Cell. Biol. 10, 2176-2181). When grown with phytosphingosine they make sphingolipids in normal amounts. SLC strains carry a secondary suppressor mutation that bypasses the need to synthesize a long chain base. Hypothetically, the suppressor mutation could function by mutating a gene whose protein product required a sphingolipid for function, by increasing the level of one or more endogenous lipids, or by making new lipid(s). Here we demonstrate that SLC strains make novel inositol glycerophospholipids when cultured without a long chain base. The novel lipids are phosphatidylinositol (PI), mannosyl-PI, and inositol-P-(mannosyl-PI), containing 1 mol of C26 fatty acid, ordinarily found in yeast sphingolipids but not usually found in glycerophospholipids; the C26 fatty acid appears to be located at the SN-2 position of the glycerol. In addition, mono-fattyacyl versions of these three compounds were also identified. The polar head groups and hydrophobic portions of these novel lipids are strikingly similar to S. cerevisiae sphingolipids found in wild-type cells. We speculate that the novel lipids structurally mimic sphingolipids and thereby compensate for some sphingolipid function(s) necessary for growth.

Chromatography, High Pressure Liquid↗