A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.

Proteins and the intracellular exchange of lipids. I. Stimulation of phospholipid exchange between mitochondria and microsomal fractions by proteins isolated from potato tuber.

1. An active fraction which stimulates the exchange of phospholipids between microsomal fractions and mitochondria was isolated by gel filtration from potato tuber homogenates. 2. This fraction is probably a protein since the stimulatory factor is nondialyzable and temperature-sensitive. The molecular weight of the active fraction is approximatively 22 000. 3. The addition of the stimulatory fraction increases essentially the exchange of the main phospholipids of the potato tuber organelles; phosphatidyl-inositol, phosphatidylcholine and phosphatidylethanolamine. 4. Inhibiting factors, probably lipid-degrading enzymes, are present in the total supernatant fraction and are responsible for the inhibiting effect on the exchange of lipids when the crude supernatant fraction is used. The "inhibition effect" is rather a degradation of the exchangeable phospholipid pools.

Cyanide sensitivity and induction of the microsomal oleoyl-CoA desaturase of potato tuber.

1. The properties of oleoyl-CoA desaturase, induced in microsomal fractions by the 'ageing' treatment of potato tuber slices (aeration of slices for 3-18 h), were investigated to study the effect of cyanide on desaturation and cycloheximide on the induction of the desaturase. 2. The electrons needed for the desaturation can be supplied either by NADH or NADPH, but ascorbate can also drive the reaction; experiments with CO suggested that cytochrome P-450 was not involved in the desaturation. 3. A strong inhibition of the desaturation by potassium cyanide was observed with each of the electron donors; total inhibition was noticed with 1 mM KCN; low concentrations (0.1 mM) caused 50% inhibition of the desaturation. 5. The variation of the oleoyl-CoA desaturase activity during the ageing process and the drop in this activity in aged slices treated by cycloheximide indicate that the enzyme undergoes an active turnover.

Purification and some properties of the squalene-tetrahymanol cyclase from Tetrahymena thermophila.

The membrane-bound enzyme from Tetrahymena thermophila responsible for the conversion of squalene into the quasi-hopanoid tetrahymanol was purified 297-fold to near homogeneity. Purification involved solubilization by octylthioglucoside, chromatography on DEAE-trisacryl, hydroxyapatite and FPLC ion-exchange on Mono Q. The apparent KM was found to be 18 microM. 2,3-Iminosqualene and N,N-dimethyldodecylamine-N-oxide are effective inhibitors of the cyclase with I50 values of 50 and 30 nM, respectively. The cyclase has a molecular mass of 72 kDa as judged by electrophoresis in polyacrylamide gels under denaturating conditions. The optimal enzymatic activity was obtained at pH 7.0 and 30 degrees C. The solubilized enzyme needs the presence of detergent for maintaining activity. The influence of different detergents on cyclase activity was studied. Triton X-100 proved to be a strong inactivator of the enzyme. Solubilization of the cyclase in Tween 80 and digitonin inactivates the enzyme. However, its activity can be recovered by complementation of the assay buffer with octylthioglucoside above its critical micellar concentration. We suggest that this approach might be applicable to other membrane-bound proteins.

Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization.

PI-synthase selectivity from etiolated maize coleptiles was studied either associated with the microsomal membranes or after solubilization by CHAPS and prepurification on a DEAE-trisacryl M column. When maize microsomes were incubated with [3H]inositol without any exogenous CPM-PA, the most heavily labelled molecular species were 16:0/18:2-PI (77%), 16:0/18:3-plus 18:2/18:2-PI (15%), 16:0/18:1-PI (4%) and 18:0/18:2-PI (4%). Addition to the incubation medium of up to 300 microM 16:0/16:0-CMP-PA unexpectedly resulted in the formation of very little labelled 16:0/16:0-PI. When the solubilized fraction from microsomes was incubated with [3H]inositol in absence of 16:0/16:0-CPM-PA, the same PI molecular species as above were synthesized. However, with increasing concentrations of 16:0/16:0-CMP-PA in the medium, increasing amounts of labelled 16:0/16:0-PI appeared as well. With prepurified PI-synthase eluted from a DEAE column, endogenous CMP-PA was poorly utilized for PI biosynthesis whereas the exogenous 16:0/16:0-CPM-PA was used actively. With time, the endogenous CMP-PA was utilized first and the exogenous substrate was utilized, albeit, much more slowly. The results demonstrate that the selectivity displayed by PI-synthase towards various molecular species of CMP-PA depends on the integration of the enzyme in the membrane structure. Solubilization of the enzyme, i.e., inclusion of the protein in micelles with detergents and lipids, results in an apparent loss of the selectivity for CMP-PA.

Control of maize lipid transfer protein activity by oxido-reducing conditions.

The activity of lipid transfer proteins (LTPs) isolated from maize, able to facilitate phospholipid movement between membranes, was studied under various oxido-reducing conditions. A progressive inactivation of LTP transfer activity was observed with increasing concentrations of reduced dithiothreitol (DTTred). This inactivation was accompanied by an increase in SH titer as well as by changes of the protein conformation deduced from its higher mobility in SDS-PAGE. By contrast, DTTred did not affect the formation of lipid-LTP complex. Transfer activity and original electrophoretic mobility were partially restored under reoxidation by air or oxidized DTT. Together, these results demonstrate the critical role of correct S-S bondings on LTP activity and suggest a possible in vivo regulation, according to the specific oxido-reducing conditions prevailing in different cell compartments.

Acyl-binding/lipid-transfer proteins from rape seedlings, a novel category of proteins interacting with lipids.

From rape (Brassica napus) seedlings proteins able to bind fatty acids and their CoA-esters were purified by gel filtration and cation-exchange chromatography. Among the four proteins detected, one of them (peak IV) appeared purified to homogeneity. This protein is a monomer with a molecular mass of about 9 kDa, as estimated by gel filtration and by polyacrylamide gel electrophoresis. The isoelectric point of the rape protein was higher than 10.5 as determined by chromatofocusing. The pure rape protein appeared furthermore to be able to transfer several phospholipids (phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine) between membranes. The rape protein, having a multifunctional property, was thus called acyl-binding/lipid-transfer protein (AB-LTP). In order to compare this protein to plant lipid-transfer proteins (LTPs), its structure was determined. The amino acid analysis of the rape AB-LTP revealed a high amount of alanine, an absence of histidine and tryptophan and the presence of eight cysteine residues. The N-terminal amino acid sequence of the rape protein revealed a high homology to plant LTPs. These observations led us to propose that the rape AB-LTPs belong to a category of plant proteins interacting with lipids and playing a role in the fatty acid dynamics.

Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis.

The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyelin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.

Use of spin-labeled and fluorescent lipids to study the activity of the phospholipid transfer protein from maize seedlings.

The transfer of spin-labeled and fluorescent lipids between sonicated vesicles and different host membranes has been measured in the presence or absence of a phospholipid transfer protein purified from maize seedlings. It was found that the protein has little specificity towards the phospholipid head group and allows the transfer of hydrophobic long chain phospholipids. By contrast, no transfer of a cholesterol analogue could be detected. By EPR spectroscopy, evidence is presented that shows that the protein catalyzes the incorporation of labeled phospholipids in the outer monolayer of the acceptor membranes. The efficiency of the transfer depends largely on the nature of the acceptor: erythrocytes are more difficult to label than chromaffin granules or liposomes made with unsaturated lipids. Thus, consistent with the high activation energy observed, the transfer is facilitated when it involves fluid membranes. These results are in favor of a process involving the exchange of phospholipids, facilitated by a shuttle protein rather than a fusion mechanism.

Biosynthesis of gamma-linolenic acid in developing seeds of borage (Borago officinalis L.).

delta 6-desaturation of [14C]linoleoyl-CoA or [14C]oleoyl-CoA leading to the synthesis of gamma-linolenic acid was studied in vitro with microsomal fractions from developing seeds of Borago officinalis. Time course of the reaction, effects of protein and precursor concentrations and nucleotide requirements were examined. These parameters allowed us to improve the in vitro delta 6-desaturation assay. We observed that the precursors were acylated mainly in phosphatidylcholine, diacylglycerol and triacylglycerol, and then desaturated. NADH was absolutely required when [14C]oleoyl-CoA was the precursor, but not when [14C]linoleoyl-CoA was the precursor although it stimulated the reaction. The in vitro delta 6-desaturase activity was found mainly in phosphatidylcholine, associated with enriched endoplasmic reticulum membranes (ER) from embryos. No activity was observed in ER from seed coat or seedling. During maturation of the seeds, delta 6-desaturase reached its highest activity 14 to 16 days after pollination.

Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

A pair of genes coding for lipid-transfer proteins in Sorghum vulgare.

Approximately five genes coding for lipid-transfer proteins (LTP) can be detected in Sorghum vulgare by DNA blots using a specific genomic probe. Two of these genes have been identified and sequenced. The two genes (ltp1 and ltp2) code for very similar (91.8% identity) proteins, they are separated by approx. 4 kb of DNA and their open reading frames may be read in the same direction. The gene (ltp1) located upstream has an intron placed in the same position already described for other ltp in maize and rice. Gene ltp2 has no intron. cDNAs corresponding to ltp1 have been identified in a 6-day-old plantlet library, but not for ltp2. The results of the comparison between the two sequences indicate the presence of a gap between the two genes in their promoter region. LTP seem to be coded for in plants by a small family of genes. At least in sorghum, two of its components are tightly clustered in the same genomic region.

Characterization of a rice gene coding for a lipid transfer protein.

The cloning and sequence analysis of a gene that encodes a lipid transfer protein (LTP) from rice is reported. A genomic DNA library from Oryza sativa was screened using a cDNA encoding a maize LTP. One genomic clone containing the gene (Ltp) was partially sequenced and analyzed. The open reading frame is interrupted by an 89-bp intron. From the results of Southern hybridizations, Ltp appears to be a member of a small multigenic family. Transcripts of the corresponding gene were detected in several tissues including coleoptile, leaf, endosperm, scutellum and root. The transcription start point was determined by primer extension. The deduced amino-acid sequence of the Ltp product is shown to be homologous to LTPs from other crops.

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing.

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.

A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

Effect of noradrenaline chronic administration on brown fat phospholipids.

Chronic cold exposure of rats (9 days at 5 degrees C) induces an alteration of the fatty acid composition of phospholipids in brown adipose tissue. The alteration is due to an increase of the unsaturation degree of these lipids. The phenomenon can be reproduced by 10(-7) mole. h-1 administration of noradrenaline for 9 days in rats kept at 25 degrees C. Thus, phospholipid alteration in brown fat of cold exposed rats is most probably a consequence of the increase of sympathetic tone which occurs in this tissue during exposure to cold.