Phosphatidylserine increase in rat liver endomembranes during the acute phase response.

The acute phase response (APR) is responsible for great changes in protein and lipid metabolism. For example, marked changes are observed in the metabolism of fatty acids, triglycerides, cholesterol and sphingolipids. Those lipids are partly recovered in the lipoproteins and subsequently in the plasma. Beside these lipid families, nothing is known about phospholipids and their synthesis in endomembranes during the APR. Our studies show that phosphatidylserine synthesis is stimulated during the APR and that this lipid is increased in the endoplasmic reticulum (ER) and the ER-derived vesicles.

Structure and expression of the murine phosphatidylserine synthase-1 gene.

In mammalian cells, phosphatidylserine is synthesized by two different enzymes, phosphatidylserine synthase (PSS)-1 and -2, via a base exchange reaction in which the head group of a phospholipid (phosphatidylcholine or phosphatidylethanolamine) is replaced by l-serine. Since the amino acid sequences of PSS1 and PSS2 are only approximately 30% identical, it is likely that they are encoded by different genes. We have screened a murine liver genomic DNA library, included in bacterial artificial chromosomes, with full-length murine PSS1 cDNA and isolated a clone containing the majority of the PSS1 gene. This gene spans approximately 35 kilobases and contains 13 exons and 12 introns. The sizes of the exons range from 44 to 1035 base pairs. The gene was localized to chromosome 13 in region B-C1. According to reverse transcriptase-mediated polymerase chain reaction, PSS1 and PSS2 mRNAs were expressed in all murine tissues examined. The mRNA encoding PSS1 was most abundant in kidney, brain, and liver, whereas PSS2 mRNA was most highly expressed in testis. In general agreement with the levels of mRNA expression, the choline exchange activity (contributed by PSS1, but not PSS2) was highest in brain, whereas serine and ethanolamine exchange activities were highest in testis and kidney. The transcriptional initiation site for PSS1 was identified 111 base pairs upstream of the ATG specifying the start of translation. The putative 5'-proximal promoter region of the gene contained no TATA or CAAT box, but did have a high GC content. Isolation of the murine PSS1 gene is a step toward generation of genetically modified mouse models that will help to understand the functions of PSS1 and PSS2 in animal biology.

One of the origins of plasma membrane phosphatidylserine in plant cells is a local synthesis by a serine exchange activity.

In plant cells, as in animal cells, the endoplasmic reticulum (ER) is considered to be the major site of phospholipid synthesis, and it has been shown that phosphatidylserine (PS) reaches the plasma membrane via the vesicular ER-Golgi-plasma membrane pathway in leek cells. However, it has never been determined whether the plasma membrane of leek cells is able to synthesize PS. We have analyzed the distribution of PS synthesizing enzymes along the vesicular pathway. In ER, Golgi and plasma membrane fractions isolated from leek cells, we have measured the activity of the two biosynthetic pathways leading to the synthesis of PS, i.e. serine exchange and CTP cytidylyltransferase plus PS synthase. We have found a high serine exchange activity in the plasma membrane fraction, and then determined that this membrane is able to synthesize both long chain fatty acid- and very long chain fatty acid-containing PS. Therefore, the PS in the plasma membrane of leek cells has two different origins: the intracellular vesicular pathway from the ER and a local synthesis in the plasma membrane.

ATP-Dependent formation of phosphatidylserine-rich vesicles from the endoplasmic reticulum of leek cells

Leek (Allium porrum) plasma membrane is enriched in phosphatidylserine (PS) by the vesicular pathway, in a way similar to that already observed in animal cells (B. Sturbois-Balcerzak, D.J. Morre, O. Loreau, J.P. Noel, P. Moreau, C. Cassagne [1995] Plant Physiol Biochem 33: 625-637). In this paper we document the formation of PS-rich small vesicles from leek endoplasmic reticulum (ER) membranes upon addition of ATP and other factors. The omission of ATP or its replacement by ATPgamma-S prevents vesicle formation. These vesicles correspond to small structures (70-80 nm) and their phospholipid composition, characterized by a PS enrichment, is compatible with a role in PS transport. Moreover, the PS enrichment over phosphatidylinositol in the ER-derived vesicles is the first example, to our knowledge, of phospholipid sorting from the ER to ER-derived vesicles in plant cells.

Transport of sterols to the plasma membrane of leek seedlings

To investigate the intracellular transport of sterols in etiolated leek (Allium porrum L.) seedlings, in vivo pulse-chase experiments with [1-14C]acetate were performed. Then, endoplasmic reticulum-, Golgi-, and plasma membrane (PM)-enriched fractions were prepared and analyzed for the radioactivity incorporated into free sterols. In leek seedlings sterols are present as a mixture in which (24R)-24-ethylcholest-5-en-3beta-ol is by far the major compound (around 60%). The other sterols are represented by cholest-5-en-3beta-ol, 24-methyl-cholest-5-en-3beta-ol, (24S)-24-ethylcholesta-5,22E-dien-3beta-ol, and stigmasta-5, 24(24(1))Z-dien-3beta-ol. These compounds are shown to reside mainly in the PM. Our results clearly indicate that free sterols are actively transported from the endoplasmic reticulum to the PM during the first 60 min of chase, with kinetics very similar to that of phosphatidylserine. Such a transport was found to be decreased at low temperature (12 degreesC) and following treatment with monensin and brefeldin A. These data are consistent with a membrane-mediated process for the intracellular transport of sterols to the PM, which likely involves the Golgi apparatus.

Antibodies to long-chain acyl-CoAs. A new tool for lipid biochemistry.

Antibodies directed against long chain acyl-CoAs (having 16 and 18 carbon atoms) have been prepared and are reported for the first time. A modified ELISA procedure adapted to these amphiphilic molecules has been developed: it is a rapid, simple and sensitive test permitting to detect as little as 3 pmol of acyl-CoA. These antibodies represent a new tool for studying long-chain acyl-CoAs. Their use in an immunochemical approach for the study of protein-acyl-CoA interactions is presented.