Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae

The incorporation of unsaturated acyl chains into phospholipids during de novo synthesis is primarily mediated by the 1-acyl-sn-glycerol-3-phosphate acyltransferase reaction. In Saccharomyces cerevisiae, Slc1 has been shown to mediate this reaction, but distinct activity remains after its removal from the genome. To identify the enzyme that mediates the remaining activity, we performed synthetic genetic array analysis using a slc1Delta strain. One of the genes identified by the screen, LPT1, was found to encode for an acyltransferase that uses a variety of lysophospholipid species, including 1-acyl-sn-glycerol-3-phosphate. Deletion of LPT1 had a minimal effect on 1-acyl-sn-glycerol-3-phosphate acyltransferase activity, but overexpression increased activity 7-fold. Deletion of LPT1 abrogated the esterification of other lysophospholipids, and overexpression increased lysophosphatidylcholine acyltransferase activity 7-fold. The majority of this activity co-purified with microsomes. To test the putative role for this enzyme in selectively incorporating unsaturated acyl chains into phospholipids in vitro, substrate concentration series experiments were performed with the four acyl-CoA species commonly found in yeast. Although the saturated palmitoyl-CoA and stearoyl-CoA showed a lower apparent Km, the monounsaturated palmitoleoyl-CoA and oleoyl-CoA showed a higher apparent Vmax. Arachidonyl-CoA, although not abundant in yeast, also had a high apparent Vmax. Pulse-labeling of lpt1Delta strains showed a 30% reduction in [3H]oleate incorporation into phosphatidylcholine only. Therefore, Lpt1p, a member of the membrane-bound o-acyltransferase gene family, seems to work in conjunction with Slc1 to mediate the incorporation of unsaturated acyl chains into the sn-2 position of phospholipids.     

Characterization of the P5 subfamily of P-type transport ATPases in mice

In mammals, the most poorly understood P-type ATPases are those of the P(5) subfamily. To begin characterization of the mammalian P(5)-ATPases, BLAST searches of DNA sequence databases were performed. Five genes were identified in the mouse, human, dog, and rat genomes, and the coding sequences of the mouse genes, termed Atp13a1-Atp13a5, were determined. The intron/exon organization of Atp13a1 differs entirely from those of Atp13a2-5, which are closely related. Amino acid sequence comparisons between the five mouse and two yeast P(5)-ATPases suggest that Atp13a1 is orthologous to the yeast Cod1 gene and that Atp13a2-5 are orthologous to yeast Yor291w. Northern blot analysis showed that Atp13a1, Atp13a2, and Atp13a3 mRNAs were expressed in all mouse tissues, whereas Atp13a4 and Atp13a5 mRNAs were restricted to brain and stomach. While the substrate specificity of these transporters is unknown, their importance is underscored by the presence of homologs in fish, insects, worms, and other eukaryotes.     

Ion transport and energy transduction of P-type ATPases: Implications from electrostatic calculations

This paper summarizes our present electrostatic calculations on P-type ATPases and their contribution to understand the molecular details of the reaction mechanisms. One focus was set on analyzing the proton countertransport of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a). Protonation of acidic residues was calculated in dependence of pH for different enzyme states in the reaction cycle of the Ca²⁺-ATPase. We proposed that the acidic Ca²⁺ ligands Glu 771, Asp 800 and Glu 908 participate in the proton countertransport whereas Glu 309 is more likely to serve as a proton shuttle between binding site I and the cytoplasm. Complementary to infrared measurements, we assigned infrared bands to specific Ca²⁺ ligands that are hydrogen bonded. Ion pathways were proposed based on the calculations and structural data. Another focus was set on analyzing the energy transduction mechanism of P-type ATPases. In accordance to electrophysiological experiments, we simulated an electric field across the membrane. The impact of the electric field was studied by an accumulated number of residue conformational and ionization changes on specific transmembrane helices. Our calculations on the Ca²⁺-ATPase and the Na⁺/K⁺-ATPase indicated that the highly conserved transmembrane helix M5 is one structural element that is likely to act as energy transduction element in P-type ATPases. Perspectives and limitations of the electrostatic calculations for future computational studies are pointed out.     

Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis

In the past several years significant advances have been made in our understanding of lecithin-cholesterol acyltransferase (LCAT) function. LCAT beneficially alters the plasma concentrations of apolipoprotein B-containing lipoproteins, as well as HDL. In addition, its proposed role in facilitating reverse cholesterol transport and modulating atherosclerosis has been demonstrated in vivo. Analysis of LCAT transgenic animals has established the importance of evaluating HDL function, as well as HDL plasma levels, to predict atherogenic risk.     

Amino acid transport and metabolism in nitrogen-starved cells of Saccharomyces cerevisiae.

Nitrogen-starved yeast derepress a general amino acid permease which transports basic and hydrophobic amino acids. Although both groups of amino acids are metabolized, the derivatives of the basic amino acids are retained by the cells, whereas those of the hydrophobic amino acids are released as acidic and neutral deaminated derivatives. The release of the deaminated derivatives of the hydrophobic amino acids only occurs in the presence of glucose, which presumably produces amino acceptors. The accumulation of intracellular amino acids results in trans-inhibition of the uptake of exogenous amino acids whether the intracellular amino acid is a basic amino acid or the product of intracellular transamination from a hydrophobic amino acid. Variation of permease and transaminase activity was measured during growth under repressed (ammonia-grown) and derepressed (proline-grown) conditions. Maximum levels for both activities occurs at the mid-exponential phase.     

Identification and characterization of the major lysophosphatidylethanolamine acyltransferase in Saccharomyces cerevisiae

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 ale1Delta 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.     

Possible role of histidine in the L-proline transport system of Saccharomyces cerevisiae

The L-proline transport system of Saccharomyces cerevisiae is shown to be specifically inactivated upon incubation of intact yeast cells with the histidine modifier diethylpyrocarbonate. The extent of inactivation is half-maximum at 0.5 mM diethylpyrocarbonate for an incubation of 2 min at 30 degrees C and pH 6.0. Under the same conditions, the time dependence of inactivation is monophasic with the second-order rate constant of 5.5 M-1 X s-1 and the maximum rate Jmax of L-proline transport is lowered by about 50%, while the KT value remains unchanged. Moreover, L-proline afforded significant protection against diethylpyrocarbonate inactivation. The complete reactivation of a partially inactivated L-proline transport system by neutral hydroxylamine and the elimination of the possibility that the modification of other amino acid residues are responsible for the inactivation, suggested that the transport protein inactivation occurs solely by a modification of histidine residues.     

Structure and function of the P-type ATPases.

The P-type ATPases are integral membrane proteins that generate essential transmembrane ion gradients in virtually all living cells. The structures of two of these have recently been elucidated at a resolution of 8 A. When considered together with the large body of biochemical information that has accrued for these transporters and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by these proteins.     

Evaluation of sterol transport from the endoplasmic reticulum to mitochondria using mitochondrially targeted bacterial sterol acyltransferase in Saccharomyces cerevisiae

To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.     

Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae

The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [³H]thymidine incorporation.     

Photoinactivation of peptide transport in Saccharomyces cerevisiae

Oligopeptides and dipeptides are transported into Saccharomyces cerevisiae by a carrier-mediated system. In the dark, leucyl-p-nitroanilide (Leu-p-NA) and leucyl-leucyl-4-azido-2-nitrophenylalanine [Leu-Leu-Phe-(4N3,2NO2)] are competitive inhibitors of peptide transport by S. cerevisiae cells. The photolysis of yeast cells in the presence of Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) at 350 nm results in an irreversible inactivation of peptide transport. Protection against this inactivation is afforded by an excess of trimethionine, a transported peptide. Photolysis with Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) does not affect amino acid or sugar transport, and cell viability is maintained throughout the irradiation procedure. A 5-min irradiation of S. cerevisiae with 2.4 microM Leu-p-NA or 15 microM Leu-Leu-Phe(4N3,2NO2) causes 50% inhibition of trimethionine uptake. p-Nitroaniline, a possible hydrolysis product generated from Leu-p-NA by cellular peptidase activity, has no effect on peptide transport. An exogenous energy source is not required for photoinactivation. The results suggest that a component(s) of the peptide transport system of S. cerevisiae is irreversibly modified by photolysis with Leu-p-NA or Leu-Leu-Phe-(4N3,2NO2) and provide the first example of the use of amino acid p-nitroanilides as photoaffinity labels.     

Glucose transport in a kinaseless Saccharomyces cerevisiae mutant.

Wild-type Saccharomyces cerevisiae organisms contain three kinases which catalyze the phosphorylation of glucose: two hexokinase isozymes (PI and PII) and one glucokinase. Glucose transport measurements for triple-kinaseless mutants, which lack all three of these kinases, confirm that the kinases are involved in the low apparent Km transport process observed in metabolizing cells. Thus kinase-positive cells containing one or more of the three kinases exhibit biphasic transport kinetics with a low apparent Km (1 to 2 mM) and high apparent Km (40 to 50 mM) component. Triple-kinaseless cells, however, exhibit only the high apparent Km component of kinase-positive cells (60 mM). Kinetic analysis of glucose transport in the triple-kinaseless cells shows that glucose is transported by a facilitated diffusion process which exhibits trans-stimulated equilibrium exchange and influx counterflow.     

Cystamine transport in spheroplasts of Saccharomyces cerevisiae

This work is the first demonstration that cystamine is actively accumulated in spheroplasts of Saccharomyces cerevisiae. We have identified and quantitatively determined the transported cystamine in extracts of spheroplasts that have been incubated over different time periods and in the presence of different amounts of cystamine. The method used, already reported in literature for the identification of natural aliphatic polyamines in biological fluids, consists of a derivatization of spheroplast extracts with dabsyl-chloride and subsequent chromatographic analysis in HPLC. Our results show that cystamine accumulation is a function of time, it increases up to 2.5 min then decreases. Transport is inhibited by natural aliphatic polyamines, which, at the same concentration of cystamine (1 mM), cause a decrease in cystamine transport of about 90% for spermidine, 50% for spermine and only 15% for putrescine. Furthermore, transport is energy-dependent as demonstrated by a significant decrease observed in the presence of 2,4-dinitrophenol, ouabain and vanadate. In particular 0.2 mM ouabain causes a decrease of more than 60% in cystamine transport. Our data suggest that cystamine is transported in Saccharomyces cerevisiae spheroplasts via the same polyamine transport system(s) known to be operating in higher eukaryotic cells.     

Functions and metabolism of sphingolipids in Saccharomyces cerevisiae

We describe recent advances in understanding sphingolipid functions and metabolism in the baker’s yeast Saccharomyces cerevisiae. One milestone has been reached in yeast sphingolipid research with the complete or nearly complete identification of genes involved in sphingolipid synthesis and breakdown. Other advances include roles for sphingolipid long-chain bases as signaling molecules that regulate growth, responses to heat stress, cell wall synthesis and repair, endocytosis and dynamics of the actin cytoskeleton. We touch briefly on other sphingolipid functions so that readers unfamiliar with the field will gain a broader view of sphingolipid research. These functions include roles in protein trafficking/exocytosis, lipid rafts or microdomains, calcium homeostasis, longevity and cellular aging, nutrient uptake, cross-talk with other lipids and the interaction of sphingolipids and antifungal drugs.