FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1976004857> ?p ?o ?g. }

• W1976004857 endingPage "28352" @default.
• W1976004857 startingPage "28344" @default.
• W1976004857 abstract "We recently demonstrated that yeast actively import lysophosphatidylethanolamine (lyso-PtdEtn) through the action of plasma membrane P-type ATPases and rapidly acylate it to form PtdEtn. The predominant lyso-PtdEtn acyltransferase (LPEAT) activity present in cellular extracts is acyl-CoA dependent, but the identity of the gene encoding this activity was unknown. We now demonstrate that a previously uncharacterized open reading frame, YOR175C, encodes the major acyl-CoA-dependent LPEAT activity in yeast and henceforth refer to it as ALE1 (acyltransferase for lyso-PtdEtn). Ale1p is an integral membrane protein and is highly enriched in the mitochondria-associated endoplasmic reticulum membrane. It is a member of the membrane-bound O-acyltransferase family and possesses a dibasic motif at its C terminus that is likely responsible for Golgi retrieval and retention in the endoplasmic reticulum. An ale1Δ strain retains only trace amounts of acyl-CoA-dependent LPEAT activity, and strains lacking the capacity for PtdEtn synthesis via the phosphatidylserine decarboxylase and Kennedy pathways show a stringent requirement for both exogenous lyso-PtdEtn and a functional ALE1 gene for viability. Ale1p catalytic activity has a pH optimum between pH 7 and 7.5 and a strong preference for unsaturated acyl-CoA substrates. We recently demonstrated that yeast actively import lysophosphatidylethanolamine (lyso-PtdEtn) through the action of plasma membrane P-type ATPases and rapidly acylate it to form PtdEtn. The predominant lyso-PtdEtn acyltransferase (LPEAT) activity present in cellular extracts is acyl-CoA dependent, but the identity of the gene encoding this activity was unknown. We now demonstrate that a previously uncharacterized open reading frame, YOR175C, encodes the major acyl-CoA-dependent LPEAT activity in yeast and henceforth refer to it as ALE1 (acyltransferase for lyso-PtdEtn). Ale1p is an integral membrane protein and is highly enriched in the mitochondria-associated endoplasmic reticulum membrane. It is a member of the membrane-bound O-acyltransferase family and possesses a dibasic motif at its C terminus that is likely responsible for Golgi retrieval and retention in the endoplasmic reticulum. An ale1Δ strain retains only trace amounts of acyl-CoA-dependent LPEAT activity, and strains lacking the capacity for PtdEtn synthesis via the phosphatidylserine decarboxylase and Kennedy pathways show a stringent requirement for both exogenous lyso-PtdEtn and a functional ALE1 gene for viability. Ale1p catalytic activity has a pH optimum between pH 7 and 7.5 and a strong preference for unsaturated acyl-CoA substrates. The yeast Saccharomyces cerevisiae has served as a valuable model system for understanding the structural and regulatory aspects of eukaryotic membrane biogenesis. As a consequence, our knowledge of the central pathways of glycerolipid synthesis, transport, and degradation in this organism is quite extensive. However, gaps still remain in the identification of genes encoding proteins that execute key enzymatic steps in yeast lipid metabolism. The lysophospholipid acyltransferases are an example of one such set of enzymes. In eukaryotic systems these activities are critical for remodeling of membrane lipids for specific purposes, such as the synthesis of dipalmitoyl-PtdCho 2The abbreviations used are: PtdChophosphatidyl (Ptd) cholineELMexogenous lysolipid metabolism pathwayEtnethanolaminelyso-PtdEtn1-acyl-2-hydroxyl-sn-glycero-3-phospho-EtnLPEATlyso-PtdEtn acyltransferaseMBOATmembrane-bound O-acyltransferaseP-Etnphosphoryl-EtnPtdInsphosphatidylinositolPtdSerphosphatidylserineERendoplasmic reticulumMAMmitochondria-associated ER membraneORFopen reading frameLPAATlyso-PtdOH acyltransferase. for lung surfactant (1Chen X. Hyatt B.A. Mucenski M.L. Mason R.J. Shannon J.M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11724-11729Crossref PubMed Scopus (154) Google Scholar, 2Nakanishi H. Shindou H. Hishikawa D. Harayama T. Ogasawara R. Suwabe A. Taguchi R. Shimizu T. J. Biol. Chem. 2006; 281: 20140-20147Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Recent reports also demonstrate that reacylation of lysolipids generated by the action of phospholipase A2 enzymes is an important mechanism in the regulation of free arachidonate levels for the production of eicosanoids in neutrophils (3Zarini S. Gijon M.A. Folco G. Murphy R.C. J. Biol. Chem. 2006; 281: 10134-10142Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Despite the importance of lysolipid acyltransferases in membrane lipid remodeling and signaling events, the identities of many of the genes encoding these activities remain unknown. In the yeast system only a lyso-PtdOH acyltransferase (Slc1p) (4Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar), an acyltransferase involved in glycosylphosphatidylinositol anchor remodeling (Gup1p) (5Bosson R. Jaquenoud M. Conzelmann A. Mol. Biol. Cell. 2006; 17: 2636-2645Crossref PubMed Scopus (116) Google Scholar), and a mitochondrial enzyme involved in cardiolipin remodeling (Taz1p) (6Testet E. Laroche-Traineau J. Noubhani A. Coulon D. Bunoust O. Camougrand N. Manon S. Lessire R. Bessoule J.J. Biochem. J. 2005; 387: 617-626Crossref PubMed Scopus (67) Google Scholar, 7Xu Y. Malhotra A. Ren M. Schlame M. J. Biol. Chem. 2006; 281: 39217-39224Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) have been unequivocally described in both a biochemical and genetic context. phosphatidyl (Ptd) choline exogenous lysolipid metabolism pathway ethanolamine 1-acyl-2-hydroxyl-sn-glycero-3-phospho-Etn lyso-PtdEtn acyltransferase membrane-bound O-acyltransferase phosphoryl-Etn phosphatidylinositol phosphatidylserine endoplasmic reticulum mitochondria-associated ER membrane open reading frame lyso-PtdOH acyltransferase. A specific lyso-PtdEtn acyltransferase (LPEAT) protein has not been identified at the molecular level from any organism. However, recent work from our laboratory has revealed a role for LPEAT as part of a new pathway for PtdEtn biosynthesis, which we henceforth refer to as the exogenous lysolipid metabolism (ELM) pathway (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In this pathway lyso-PtdEtn is imported by P-type ATPases at the plasma membrane followed by trafficking of this lipid to the site(s) of LPEAT action. The PtdEtn, thus, formed is capable of fully supplying both the structural requirements of the mitochondria for respiratory growth and the structural and biosynthetic requirements of the endoplasmic reticulum for PtdCho synthesis. Furthermore, the flux through the ELM pathway and quantities of PtdEtn and PtdCho thus produced are sufficient to satisfy the requirements of rapidly dividing cells for membrane biogenesis, even in the absence of all other PtdEtn synthesis pathways. In this report we have focused upon 1) identifying the gene encoding the major LPEAT in yeast, 2) characterizing the biochemical properties of the enzyme, 3) determining the subcellular localization of the enzyme, and 4) elucidating the conditions under which the LPEAT activity is essential for growth. We now present data showing that an uncharacterized yeast gene, YOR175C, which we name ALE1, encodes the major LPEAT activity in yeast. Deletion of this gene abolished essentially all LPEAT activity in cell extracts. Ale1p enzymatic activity is enriched in the mitochondria-associated ER membrane (MAM), and the kinetic properties of this enzyme led to the preferential placement of an unsaturated fatty acid at the sn-2 position of lyso-PtdEtn. The LPEAT activity of Ale1p is also required for efficient Kennedy pathway-independent utilization of lyso-PtdEtn. Materials—Unless otherwise noted, all chemicals, solvents, and amino acids for media were purchased from Sigma or Fisher. Yeast extract, peptone, and yeast nitrogen base were from Difco. Silica-60 TLC plates were from EM Sciences. All lipids were purchased from Avanti Polar Lipids (Alabaster, AL), except acyl-CoA substrates, which were from Sigma. Lyso-Ptd-Etn (Avanti) was purchased as a 20 mg/ml chloroform solution, and this solvent was removed under vacuum followed by repeated addition and evaporation of methanol to eliminate traces of chloroform. The dried lyso-PtdEtn was dissolved in 10% (v/v) Tergitol Nonidet P-40, filter-sterilized, and stored at -20 °C until use. Synthesis of radiolabeled lyso-PtdEtn was as previously described (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Yeast Culture and Genetic Manipulations—Yeast strains and their associated genotypes are provided in Tables 1 and 2. Yeast strains with deletions of specific open reading frames (ORFs) were constructed by standard methods involving one-step gene replacement (9Gietz R.D. Woods R.A. Methods Enzymol. 2002; 350: 87-96Crossref PubMed Scopus (2099) Google Scholar). Gene replacements were carried out by transformation of PCR fragments containing an appropriate marker gene (as indicated in Table 1) flanked by at least 40 base pairs of DNA identical to the 5′ and 3′ regions outside the start and stop codons of the ORF of interest. The eviction of target genes from the resultant drug-resistant or prototrophic colonies was confirmed by PCR amplification of the 5′ and 3′ recombination junctions using appropriate combinations of marker gene and target gene-specific primers. Strains were routinely maintained on standard 1% yeast extract, 2% peptone (YP) medium containing either 2% lactate (YPL) or 2% glucose (YPD) as a carbon source with the addition of 40 mg/liter adenine, 40 mg/liter uracil, and 2 mm Etn (YPDAUE or YPLAUE media). For media containing lyso-PtdEtn, 1% (v/v) Tergitol Nonidet P-40 was included, and lyso-PtdEtn was added to the desired concentration from a sterile 25 mm stock solution in 10% (v/v) Tergitol Nonidet P-40. In some experiments defined media were used consisting of yeast nitrogen base (Difco) at 6.7 g/liter, complete amino acid mix, and either glucose to give SCG media or lactate to give SCL. In the lysolipid uptake experiments, 1% Tergitol Nonidet P-40 was included, and this medium is denoted SCGT. For determination of growth requirements, 5-fold serial dilutions of yeast cultures were spotted onto plates as described previously (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).TABLE 1Strains used for directed genetic screening for defects in LPEAT activity All strains were generated as part of a genome-wide functional analysis study (26Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Liebundguth N. Lockhart D.J. Lucau-Danila A. Lussier M. M'Rabet N. Menard P. Mittmann M. Pai C. Rebischung C. Revuelta J.L. Riles L. Roberts C.J. Ross-MacDonald P. Scherens B. Snyder M. Sookhai-Mahadeo S. Storms R.K. Veronneau S. Voet M. Volckaert G. Ward T.R. Wysocki R. Yen G.S. Yu K. Zimmermann K. Philippsen P. Johnston M. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3210) Google Scholar) and were purchased from Open Biosystems (Huntsville, AL).StrainGenotypeBY4742Mat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0ybr042cΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ybr042cΔ::KANRypr140wΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 taz1Δ::KANRyor298wΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 mum3Δ::KANRydl052cΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 slc1Δ::KANRydr018cΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 dr018cΔ::KANRygl084cΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 gup1Δ::KANRypl189wΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 gup2Δ::KANRyor175cΔMat α his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ale1Δ::KANR Open table in a new tab TABLE 2Strains used for functional characterization of Ale1pStrainGenotypeSourceSEY6210Mat α trp1 leu2 ura3 lys2 his3 suc2Scott Emr, Univ. of California, San DiegoPTY44Mat α trp1 leu2 ura3 lys2 his3 suc2 psd1Δ::TRP1 psd2Δ::HIS327WRY8Mat α trp1 leu2 ura3 lys2 his3 suc2 psd1Δ::TRP1 psd2Δ::HIS3 ect1Δ::KANR8JWY89Mat α trp1 leu2 ura3 lys2 his3 suc2 psd1Δ::TRP1 psd2Δ::HIS3 ale1Δ::HYGRThis studyWRY90Mat α trp1 leu2 ura3 lys2 his3 suc2 psd1Δ::TRP1 psd2Δ::HIS3 ale1Δ::HYGR ect1Δ::KANRThis studyWRY28Mat α trp1 leu2 ura3 lys2 his3 suc2 psd1Δ::TRP1 psd2Δ::HIS3 lem3Δ:: KANR8 Open table in a new tab Directed Screening of Candidate Acyltransferase Mutants for Loss of LPEAT Activity—We identified candidate ORFs in the yeast genome encoding known and putative acyltransferase enzymes (selection of candidates is described under “Results”) and screened their cognate deletion strains (Table 1) for LPEAT activity. Culture of these mutant strains, preparation of homogenates, and LPEAT assays were conducted as previously described (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Labeling of Wild-type and ale1Δ Mutant Cells with Radioactive Lyso-PtdEtn—We carried out lyso-PtdEtn supplementation studies with strains PTY44 (ALE1) and JWY89 (ale1Δ). The strains were initially grown overnight in 5 ml of YPDAUE, and a 2-ml aliquot of culture was diluted into 50 ml of fresh YPDAUE medium. Upon reaching an A600 of 0.5, the cultures were centrifuged, and the cells were washed twice with SCGT media and suspended in 10 ml of SCGT (final A600 = 2.5). 2 ml of this cell preparation was incubated with 100,000 cpm of 1-oleoyl-2-hydroxyl-3-sn-glycerophospho-[U-14C]Etn (diluted with unlabeled compound to 250 μm) for 2 h. The cells were recovered by centrifugation and washed twice in SCGT and once in water. An aliquot of the washed cells was taken for liquid scintillation spectrometry before lipid extraction to determine the total amount of radioactivity taken up by the cells. Another aliquot of the cells was subjected to lipid extraction by the addition of 200 μl of water and 300 μl of ethanol in a sealed tube, incubation in a boiling water bath for 45 min, and followed by the addition of 4 ml of chloroform/methanol (1:1, v/v) and 1.6 ml of water. The tubes were vigorously mixed and centrifuged to separate the phases, and the upper aqueous phase was removed to a separate tube and lyophilized. This material was dissolved in 0.2 ml of water, and the radioactivity of an aliquot of the aqueous phase was measured by liquid scintillation spectrometry. The remainder of this aqueous fraction was resolved by TLC on Silica 60 plates (EM Sciences) in the solvent 0.5% (w/v) aqueous NaCl, ethanol, n-butanol, 28% ammonium hydroxide (10:5:5:1, v/v/v/v). Excess unlabeled Etn, phosphoethanolamine (P-Etn), CDP-Etn, and sn-glycero-3-P-Etn were added as carrier to facilitate the identification of radiolabeled compounds, and the bands were identified by staining with ninhydrin reagent (0.2% w/v in acetone.) The radioactive products in the organic phase were resolved by Silica-60 TLC in the system chloroform, methanol, isopropanol, 0.25% (w/v) aqueous KCl, triethylamine (30:9:25:6:18, v/v/v/v/v) and quantified by liquid scintillation spectrometry. Cell Fractionation and Enzyme Assays—Mitochondria, MAM, and microsomes were isolated by published methods (10Glick B.S. Pon L.A. Methods Enzymol. 1995; 260: 213-223Crossref PubMed Scopus (287) Google Scholar) from cultures of the appropriate strains (1.5 liters, final A600 = 0.5–0.6). The protein concentration of the subcellular fractions was measured using Bradford reagent (Bio-Rad). Ptd-Ser synthase and PtdIns synthase activities were determined by published methods (11Carman G.M. Bae-Lee M. Methods Enzymol. 1992; 209: 298-305Crossref PubMed Scopus (27) Google Scholar, 12Carman G.M. Fischl A.S. Methods Enzymol. 1992; 209: 305-312Crossref PubMed Scopus (40) Google Scholar). Acyltransferase activity was determined in 200-μl reactions containing 50 mm HEPES, pH 6.0, 1 mm EDTA, 150 mm NaCl, 100 μm 1-oleoyl-2-hydroxyl-3-sn-glycerophospho-[U-14C]Etn (5 × 104 cpm), 0–20 μm acyl-CoA, and 5–20 μg of protein from the appropriate subcellular fraction. Assays were conducted for 10 min at 30 °C, and the reactions were stopped by the addition of 250 μl of methanol and 250 μl of chloroform followed by vigorous vortexing and centrifugation in a microcentrifuge tube to separate the phases. Radioactive PtdEtn was identified by thin-layer chromatography on silica-60 TLC plates in the solvent system chloroform/methanol/water (65/25/4, v/v/v) and quantified as described above. Lyso-PtdOH acyltransferase assays were conducted using the same buffer compositions as for the LPEAT, with 100 μm lyso-PtdOH (Avanti) and 100,000 cpm of 1-[14C]oleoyl-CoA (American Radiolabeled Chemicals). LPEAT Kinetic Assays—Data for pH dependence and acyl-CoA kinetics were generated using a 100,000 × g microsomal membrane fraction isolated from PTY44 cells after lysis by bead-beating in a Bio-Spec apparatus as described previously (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Homogenization buffer and assay conditions were the same as those used for the determination of LPEAT activity in subcellular fractions. Calculation of the Km and Vmax for the acyl-CoA substrates was carried out by direct fitting of a hyperbola to the kinetic data with the program Hyper. YOR175C (ALE1) Encodes the Major Lyso-PtdEtn Acyltransferase Activity in Yeast—Our recent work examining the uptake and metabolism of lyso-PtdEtn by yeast (8Riekhof W.R. Voelker D.R. J. Biol. Chem. 2006; 281: 36588-36596Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) prompted us to investigate the acyl-CoA-dependent LPEAT activity of yeast cells. We took a reverse-genetic, candidate-gene-based approach toward finding the gene(s) specifying the LPEAT activity. Our candidate genes were selected on the basis of similarity to the known acyltransferases Taz1p, Gup1p, Slc1p, Dga1p, and Are1p, and the complete list of knock-out strains and corresponding ORF designations that were tested for loss of LPEAT activity are listed in Table 1. Homogenates of these yeast strains harboring deletions in the candidate ORFs were tested for LPEAT activity, and as shown in Fig. 1, deletion of the uncharacterized membrane-bound O-acyltransferase (MBOAT) family member, YOR175C produces a >95% depletion in the LPEAT activity of the homogenate relative to the wild-type BY4742 strain. All of the other candidates we tested had LPEAT activities comparable with the wild-type (not shown). This led us to designate the YOR175C gene as ALE1 due to its activity as an acyltransferase for lyso-PtdEtn. We next constructed a strain for functional analysis of the ALE1 gene by introducing an ale1Δ allele into the PTY44 background (psd1Δ psd2Δ) to give strain JWY89 (psd1Δ psd2Δ ale1Δ). The LPEAT activity of PTY44 was essentially identical to that of BY4742 (Fig. 1), and as with the BY4742 background, deletion of the ALE1 gene in the psd1Δ psd2Δ mutant essentially abolished the LPEAT activity of the extract. This series of experiments also revealed that, when an acyl-CoA generating system is used to drive the reaction, the SEY6210 wild-type background has a 2–3-fold higher LPEAT-specific activity relative to the BY4742 background and that the PTY44 (psd1Δ psd2Δ) background was slightly higher still (data not shown), perhaps indicating an increased expression of acyl-CoA synthetase activity. For this reason, further experiments were conducted in strains derived from the PTY44 background, except where noted. PTY44 also serves as the Etn/lyso-PtdEtn auxotrophic background for the genetic and physiological studies of lyso-PtdEtn metabolism that are described below. In Silico and Genome-scale Analyses of Ale1p—After establishing that YOR175C (ALE1) encodes the major LPEAT activity in yeast, we examined its primary sequence using various prediction algorithms to gain information about its putative localization and function. TargetP (13Emanuelsson O. Nielsen H. Brunak S. von Heijne G. J. Mol. Biol. 2000; 300: 1005-1016Crossref PubMed Scopus (3638) Google Scholar) failed to predict a secretory signal peptide or a mitochondrial targeting sequence; however, the genome-scale GFP tagging localization analysis of Huh et al. (14Huh W.K. Falvo J.V. Gerke L.C. Carroll A.S. Howson R.W. Weissman J.S. O'Shea E.K. Nature. 2003; 425: 686-691Crossref PubMed Scopus (3325) Google Scholar) indicated that Ale1p is localized to the endoplasmic reticulum. This preliminary localization analysis was corroborated by the highly sensitive TM-HMM algorithm that predicts at least seven transmembrane helices and by the presence of a di-lysine motif at the C terminus of Ale1p, which typically acts as a Golgi-retrieval and ER retention signal for membrane proteins through interaction with the coatomer complex (15Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (484) Google Scholar). These data as well as a multiple sequence alignment with other acyltransferases are given in Fig. 2. Additional evidence regarding the function of Ale1p comes from the epistatic mini-array profiling data of Schuldiner et al. (16Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. Weissman J.S. Krogan N.J. Cell. 2005; 123: 507-519Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar). This study detected a profound growth defect in the ale1Δ slc1Δ double mutant, the implications of which are described below. Biochemical Properties of ALE1—We characterized the basic biochemical and kinetic parameters of the enzyme with lyso-PtdEtn as substrate, including a pH versus activity profile and determination of the Km and Vmax for various acyl-CoA substrates. As shown in Fig. 3A, the enzymatic activity is relatively insensitive to changes in pH, and the rate versus pH profile shows a broad maximum between 6.5 and 7.5. We next examined the acyl-CoA substrate preference of Ale1p by conducting kinetic assays with various acyl-CoA species. We determined the Km and Vmax for the acyl donors by varying the substrate concentrations and measuring initial rates of the conversion of lyso-PtdEtn to PtdEtn using the most active microsomal fraction as a source of enzyme. We examined oleoyl (18:1)-, palmitoleoyl (16:1)-, palmitoyl (16:0)-, and myristoyl (14:0)-CoA substrates, given that these are the most abundant fatty-acyl species in yeast phopholipids (17Choi J.Y. Stukey J. Hwang S.Y. Martin C.E. J. Biol. Chem. 1996; 271: 3581-3589Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). As shown in Fig. 3B, the Km values for the saturated acyl-CoA substrates were significantly lower than the unsaturated substrates. For palmitoyl- and myristoyl-CoA, these values were 0.9 and 0.4 μm, respectively, and for oleoyl- and palmitoleoyl-CoA species, the Km values were 10 and 17 μm, respectively. Conversely, the Vmax values were much higher for the unsaturated species (38 and 44 nmol/min/mg of protein for oleoyl- and palmitoleoyl-CoA versus 3.7 and 1.2 nmol/min/mg of protein for palmitoyl- and myristoyl-CoA. The figure clearly shows that, at any given acyl-CoA concentration, the initial rate with the unsaturated substrates was significantly higher than the saturated substrates. Although the specificity constant (Km/Vmax) for all substrates was similar, the kinetic data clearly indicate that, given equivalent concentrations of the different acyl-CoA species, Ale1p preferentially esterifies an unsaturated acyl chain at the sn-2 position of lyso-PtdEtn. Ale1p Acts as the Major Lyso-PtdOH Acyltransferase in Yeast—The product of the SLC1 gene has been shown to act as a lyso-PtdOH acyltransferase (LPAAT) (4Nagiec M.M. Wells G.B. Lester R.L. Dickson R.C. J. Biol. Chem. 1993; 268: 22156-22163Abstract Full Text PDF PubMed Google Scholar, 18Athenstaedt K. Daum G. J. Bacteriol. 1997; 179: 7611-7616Crossref PubMed Scopus (98) Google Scholar). However, haploid slc1Δ mutants are viable, which necessitates that another, redundant LPAAT activity be present in the cell. The epistatic mini-array profile of Schuldiner et al. (16Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. Weissman J.S. Krogan N.J. Cell. 2005; 123: 507-519Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar) revealed a synthetic-lethal interaction between the ale1Δ and slc1Δ mutations. One explanation for this result is that Ale1p provides the redundant lyso-PtdOH acyltransferase activity in the slc1Δ mutant, thus rendering the double mutant incapable of making PtdOH. To test this hypothesis, we assayed LPAAT activity in the BY4742 wild type and the isogenic slc1Δ and ale1Δ mutants. As shown in Fig. 4, relative to the wild-type parent, the ale1Δ mutant shows a ∼85% decrease in the total LPAAT activity of a cellular homogenate. Surprisingly, we detected no loss of LPAAT activity in the slc1Δ mutant, which may indicate that the Ale1p LPAAT activity is up-regulated in the absence of the Slc1p activity. Taken together with the synthetic lethality data (16Schuldiner M. Collins S.R. Thompson N.J. Denic V. Bhamidipati A. Punna T. Ihmels J. Andrews B. Boone C. Greenblatt J.F. Weissman J.S. Krogan N.J. Cell. 2005; 123: 507-519Abstract Full Text Full Text PDF PubMed Scopus (694) Google Scholar), this result strongly suggests that Ale1p and Slc1p act as redundant LPAAT activities, with Ale1p accounting for the majority of the catalytic units. Subcellular Distribution of Ale1p Activity—The TM-HMM prediction described above indicated the presence of at least seven transmembrane helices, indicating an extremely high probability that Ale1p is a membrane-bound enzyme. Analysis with the localization prediction algorithm TargetP (13Emanuelsson O. Nielsen H. Brunak S. von Heijne G. J. Mol. Biol. 2000; 300: 1005-1016Crossref PubMed Scopus (3638) Google Scholar) indicated the lack of either a canonical mitochondrial or secretory targeting signal, whereas the presence of a C-terminal di-basic motif was indicative of an endoplasmic reticulum protein (15Cosson P. Letourneur F. Science. 1994; 263: 1629-1631Crossref PubMed Scopus (484) Google Scholar). These results suggested that Ale1p activity might be associated with an ER-derived microsomal fraction. Given the ability of PtdEtn derived from lyso-PtdEtn to preferentially replenish the mitochondrial PtdEtn pool, we also entertained the possibility that Ale1p, like some other enzymes of phospholipid biosynthesis, would be enriched in the ER-derived MAM. To test this hypothesis, we prepared sucrose-gradient-purified mitochondria, MAM, microsomes, and cytosol and measured the specific activity of the Ale1p LPEAT in these fractions. Data from fractions isolated from isogenic wild-type and ale1Δ mutant strains were compared, and in these purified fractions the LPEAT activity of the ale1Δ mutant was <2% that of the wild-type level. This level of activity is at the limit of detection under our assay conditions; hence, only data for fractions from the wild-type strain are given. Fig. 5C shows that the MAM fraction is the most highly enriched for LPEAT activity and gives 2–3-fold higher specific activity than the microsomal and mitochondrial fractions, which are similar in their LPEAT specific activity. Essentially no activity was detectable in the cytosolic fraction. To verify the purity of the respective preparations, the purified membrane fractions were also assayed for PtdSer and PtdIns synthase activities, and these results are also presented in Fig. 5. PtdSer and PtdIns synthase activities are known to be enriched in the MAM fraction (19Gaigg B. Simbeni R. Hrastnik C. Paltauf F. Daum G. Biochim. Biophys. Acta. 1995; 1234: 214-220Crossref PubMed Scopus (168) Google Scholar), with the remaining activity present in microsomes and only traces of activity in gradient-purified mitochondria. Our data recapitulate these published activity profiles for PtdSer and PtdIns synthases (Fig. 5, A and B) and, thus, establish the relative purity of our subcellular fractions. Given that there is substantial LPEAT activity in the purified mitochondrial fraction, the PtdSer and PtdIns synthase data also show that the Ale1p LPEAT activity that we detect in the mitochondrial fraction does not arise from contaminating microsomes. This fact provides strong evidence that the Ale1p enzyme is preferentially localized in a subfraction of the MAM that is tightly bound to the mitochondrial outer membrane such that it co-purifies with mitochondria on sucrose gradients. In the Absence of Ale1p, Lyso-PtdEtn Is Degraded to Water-soluble Products—The data presented in Fig. 6 demonstrate the catabolism of lyso-PtdEtn in the absence of Ale1p. PtdSer decarboxylase-deficient yeast strains either containing (PTY44) or lacking (JWY89) the Ale1p acyltransferase were incubated for 2 h with labeled lyso-Ptd[14C]Etn. The cells were harvested and washed extensively followed by lipid extraction and partitioning of the radiolabeled products between aqueous and organic phases. As shown in Fig. 6, A and B, the ale1Δ mutation results in a 4–5-fold higher proportion of the imported label being present in the aqueous fraction. For the ALE1 wild-type strain, only about 6–7% of the total label associated with the cells was present in the aqueous fraction compared with ∼30–35% in the ale1Δ mutant. These findings are consistent with lyso-PtdEtn entering either of two pathways after internalization (see Fig. 8); s" @default.
• W1976004857 created "2016-06-24" @default.
• W1976004857 creator A5014768508 @default.
• W1976004857 creator A5016720043 @default.
• W1976004857 creator A5017828199 @default.
• W1976004857 creator A5044700206 @default.
• W1976004857 date "2007-09-01" @default.
• W1976004857 modified "2023-10-01" @default.
• W1976004857 title "Identification and Characterization of the Major Lysophosphatidylethanolamine Acyltransferase in Saccharomyces cerevisiae" @default.
• W1976004857 cites W1424883175 @default.
• W1976004857 cites W1483456869 @default.
• W1976004857 cites W1509952659 @default.
• W1976004857 cites W1588290914 @default.
• W1976004857 cites W1595781572 @default.
• W1976004857 cites W1598208661 @default.
• W1976004857 cites W1905318621 @default.
• W1976004857 cites W1967042190 @default.
• W1976004857 cites W1971388047 @default.
• W1976004857 cites W1977911448 @default.
• W1976004857 cites W1982268890 @default.
• W1976004857 cites W2017550994 @default.
• W1976004857 cites W2024039941 @default.
• W1976004857 cites W2028123717 @default.
• W1976004857 cites W2042688172 @default.
• W1976004857 cites W2043513653 @default.
• W1976004857 cites W2054593368 @default.
• W1976004857 cites W2061437688 @default.
• W1976004857 cites W2062286749 @default.
• W1976004857 cites W2071941416 @default.
• W1976004857 cites W2111592695 @default.
• W1976004857 cites W2112727465 @default.
• W1976004857 cites W2118253143 @default.
• W1976004857 cites W2140948740 @default.
• W1976004857 cites W2152458080 @default.
• W1976004857 cites W2169805130 @default.
• W1976004857 cites W2410585811 @default.
• W1976004857 doi "https://doi.org/10.1074/jbc.m705256200" @default.
• W1976004857 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17652094" @default.
• W1976004857 hasPublicationYear "2007" @default.
• W1976004857 type Work @default.
• W1976004857 sameAs 1976004857 @default.
• W1976004857 citedByCount "146" @default.
• W1976004857 countsByYear W19760048572012 @default.
• W1976004857 countsByYear W19760048572013 @default.
• W1976004857 countsByYear W19760048572014 @default.
• W1976004857 countsByYear W19760048572015 @default.
• W1976004857 countsByYear W19760048572016 @default.
• W1976004857 countsByYear W19760048572017 @default.
• W1976004857 countsByYear W19760048572018 @default.
• W1976004857 countsByYear W19760048572019 @default.
• W1976004857 countsByYear W19760048572020 @default.
• W1976004857 countsByYear W19760048572021 @default.
• W1976004857 countsByYear W19760048572022 @default.
• W1976004857 countsByYear W19760048572023 @default.
• W1976004857 crossrefType "journal-article" @default.
• W1976004857 hasAuthorship W1976004857A5014768508 @default.
• W1976004857 hasAuthorship W1976004857A5016720043 @default.
• W1976004857 hasAuthorship W1976004857A5017828199 @default.
• W1976004857 hasAuthorship W1976004857A5044700206 @default.
• W1976004857 hasBestOaLocation W19760048571 @default.
• W1976004857 hasConcept C104317684 @default.
• W1976004857 hasConcept C116834253 @default.
• W1976004857 hasConcept C185592680 @default.
• W1976004857 hasConcept C2775970874 @default.
• W1976004857 hasConcept C2776330855 @default.
• W1976004857 hasConcept C2777576037 @default.
• W1976004857 hasConcept C2778918659 @default.
• W1976004857 hasConcept C2779222958 @default.
• W1976004857 hasConcept C2781112429 @default.
• W1976004857 hasConcept C41625074 @default.
• W1976004857 hasConcept C55493867 @default.
• W1976004857 hasConcept C59822182 @default.
• W1976004857 hasConcept C70721500 @default.
• W1976004857 hasConcept C86803240 @default.
• W1976004857 hasConceptScore W1976004857C104317684 @default.
• W1976004857 hasConceptScore W1976004857C116834253 @default.
• W1976004857 hasConceptScore W1976004857C185592680 @default.
• W1976004857 hasConceptScore W1976004857C2775970874 @default.
• W1976004857 hasConceptScore W1976004857C2776330855 @default.
• W1976004857 hasConceptScore W1976004857C2777576037 @default.
• W1976004857 hasConceptScore W1976004857C2778918659 @default.
• W1976004857 hasConceptScore W1976004857C2779222958 @default.
• W1976004857 hasConceptScore W1976004857C2781112429 @default.
• W1976004857 hasConceptScore W1976004857C41625074 @default.
• W1976004857 hasConceptScore W1976004857C55493867 @default.
• W1976004857 hasConceptScore W1976004857C59822182 @default.
• W1976004857 hasConceptScore W1976004857C70721500 @default.
• W1976004857 hasConceptScore W1976004857C86803240 @default.
• W1976004857 hasIssue "39" @default.
• W1976004857 hasLocation W19760048571 @default.
• W1976004857 hasOpenAccess W1976004857 @default.
• W1976004857 hasPrimaryLocation W19760048571 @default.
• W1976004857 hasRelatedWork W1982415357 @default.
• W1976004857 hasRelatedWork W2020624253 @default.
• W1976004857 hasRelatedWork W2046585160 @default.
• W1976004857 hasRelatedWork W2048891172 @default.
• W1976004857 hasRelatedWork W2080092217 @default.
• W1976004857 hasRelatedWork W2113204264 @default.

• next