Sterol trafficking between the endoplasmic reticulum and plasma membrane in yeast.

We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816-5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154-158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.

Cell surface display and intracellular trafficking of free glycosylphosphatidylinositols in mammalian cells.

In addition to serving as membrane anchors for cell surface proteins, glycosylphosphatidylinositols (GPIs) can be found abundantly as free glycolipids in mammalian cells. In this study we analyze the subcellular distribution and intracellular transport of metabolically radiolabeled GPIs in three different cell lines. We use a variety of membrane isolation techniques (subcellular fractionation, plasma membrane vesiculation to isolate pure plasma membrane fractions, and enveloped viruses to sample cellular membranes) to provide direct evidence that free GPIs are not confined to their site of synthesis, the endoplasmic reticulum, but can redistribute to populate other subcellular organelles. Over short labeling periods (2.5 h), radiolabeled GPIs were found at similar concentration in all subcellular fractions with the exception of a mitochondria-enriched fraction where GPI concentration was low. Pulse-chase experiments over extended chase periods showed that although the total amount of cellular radiolabeled GPIs decreased, the plasma membrane complement of labeled GPIs increased. GPIs at the plasma membrane were found to populate primarily the exoplasmic leaflet as detected using periodate oxidation of the cell surface. Transport of GPIs to the cell surface was inhibited by Brefeldin A and blocked at 15 degrees C, suggesting that GPIs are transported to the plasma membrane via a vesicular mechanism. The rate of transport of radiolabeled GPIs to the cell surface was found to be comparable with the rate of secretion of newly synthesized soluble proteins destined for the extracellular space.

Segregation of glycosylphosphatidylinositol biosynthetic reactions in a subcompartment of the endoplasmic reticulum.

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.

Pharmacological and molecular properties of opioid binding sites synthesized in a cell-free translation system.

Cell-free translation of mRNA, extracted from NG108-15 cells, was used to examine some properties of the opioid binding sites synthesized in vitro. A monoclonal antiidiotype antibody directed against the delta opioid receptor immunoprecipitated a major band of Mr 51,000. Translational immunoassays of poly[A]+RNA, size fractionated by methylmercury agarose gel electrophoresis, demonstrated that the 51,000 Mr protein specifically immunoprecipitated by the anti-opioid receptor antiidiotype antibodies was coded by a transcript which length was in the 6 to 8 kb range. Displacement binding studies of tritiated ligands (either bremazocine or delta or mu selective peptides) with type selective opioid ligands showed that only one type of opioid binding site was synthesized in vitro. Although the pharmacological profiles of ligands binding to NG108-15 cells were characteristic of the delta receptor type, the de novo synthesized opioid binding site had lost its delta selectivity and showed equal affinity for both the mu and delta but not for the kappa ligands. Similar to our finding using the immunoprecipitation system, size fractionation of the NG108-15 poly[A]+RNA demonstrated that the transcript coding for the "mu-delta" binding site had a length of 6,500 to 7,500 nucleotides.

Tamm-Horsfall protein, a kidney marker is expressed on brain sulfogalactosylceramide-positive astroglial structures.

The Tamm-Horsfall (TH) glycoprotein and the acidic glycosphingolipid sulfogalactosylceramide (SGC) have a strictly superimposable localization on kidney tissue sections. The fact that SGC is a prevalent glycolipid in mammalian brain, prompted us to look for the presence of TH in the rat central nervous system (CNS). An antiserum raised against human TH was found to react with rat CNS homogenate in the complement fixation assay. This anti-TH antiserum recognized a rat CNS protein having an identical electrophoretical mobility on SDS polyacrylamide gel electrophoresis (PAGE). Indirect immunofluorescence on rat brain tissue sections allowed us to localize this brain TH cross-reacting material to ependymal cells and astrocytic processes such as the Bergmann fibers or astrocytic feet in contact with either the blood vessels or the meninges. All these astroglial structures are also SGC-positive. Since TH and SGC in the kidney are localized on a membrane that possesses an electrogenic Cl-pump, we propose that the astroglial structures which contain these two molecules are also the site of a Cl-transport system.

Immunohistochemical localization of galactosyl and sulfogalactosyl ceramide in the brain of the 30-day-old mouse.

We have used purified antibodies against galactosylceramide (galCb) and sulfogalactosylceramide (sulf) to study the topographical distribution of these two lipid haptens in the brain of the 30-day-old mouse. This study has been conducted, using the indirect immunofluorescence method, on cerebellum, brain stem and hemispherical tissue sections. Both haptens are present in the myelin sheaths and in the oligodendrocytes within the myelinated bundles. Cortical oligodendrocytes as well as some of the subependymal cells are also galCb-positive but sulf-negative. On the contrary, ciliated ependymal cells and subpial astrocytic processes (especially the Bergmann glia fibers in the cerebellum) are sulf-positive and galCb-negative. Astrocyte cell bodies and other astrocytic cell processes are devoid of both haptens. Lastly, some-sulf positive galCb-negative processes, as yet unidentified, were also found in the periaqueductal gray matter and in the nucleus interpeduncularis.

Pelizaeus-Merzbacher disease: biochemical analysis of isolated myelin (electron-microscopy; protein, lipid and unsubstituted fatty acids analysis).

Analysis of myelin from a leukodystrophic brain was performed (Pelizaeus-Merzbacher disease, classical type). Myelin recovery was 7% of normal, when isolated by ultracentrifugation. Electron microscopy showed a great amount of loose lamellae, with less thick sheaths and periodicity close to normal. This myelin contains fewer lipids than normal, sphingolipids and plasmalogens being reduced. FAtty acids from phospholipids are essentially normal, however enols from plasmalogens are largely reduced. Purified sphingolipids (cerebrosides, sulfatides and sphingomyelin) present a considerable diminution in very long chain fatty acids; the ratio of very long chain fatty acids (over C18) on shorter chains is 1% of the normal value for saturated fatty acids and 2% for the monounsaturated homologues. Protein analysis showed that basic protein and proteolipids were reduced, Wolfgram proteins being relatively increased.

Determination of glial fibrillary acidic protein (GFAP) in human brain tumors.

Brain tumors have been tested for their glial fibrillary acidic protein (GFAP) content by means of the rocket electrophoresis technique. Meningiomas and neurinomas were low in GFAP. Metastases had a low level of GFAP except when contaminated with surrounding tissue. Non-nervous tumors such as myeloma, myeloplaxoma and adenocarcinoma gave negative results. More detailed correlations with histological observations have been looked for in glial tumors. Low levels of GFAP were always associated with signs of malignancy such as mitoses and giant or atypical cells, whereas high levels of GFAP were correlated with the presence of well-preserved astrocytes.

Lignoceric acid biosynthesis in the developing brain. Activities of mitochondrial acetyl-CoA-dependent synthesis and microsomal malonyl-CoA chain-elongating system in relation to myelination. Comparison between normal mouse and dysmyelinating mutants (quaking and jimpy).

Age-related changes in the activities of microsomal and mitochondrial elongating systems have been determined in mouse brain from birth to maturity. In microsomes, the components necessary for behenyl-CoA (docosanoly-CoA) elongation have been found to be NADPH and malonyl-CoA. In mitochondria, both NADH and NADPH are used and acetyl-CoA is the only donor of two-carbon-atoms unit. The synthesised fatty acids were identified by thin-layer and gas chromatography. The specific activity is higher in microsomes than in mitochondria. In microsomes, the specific activity for malonyl-Co-A incorporation reached a maximum at 15 - 20 days of age; this peak was not obtained in the Quaking and Jimpy mutants. The increase in enzyme activity (specific activity and total activity per brain) paralleled the myelin deposition. The activity of the mitochondrial system increases regularly during development: it is not correlated to myelination and it is not affected in the Quaking mutant. The interplay between microsomal and mitochondrial elongation systems is studied.

Mouse brain uptake and metabolism of stearic acid.

After injection, labelled stearic acid is transported directly into the brain and incorporated into brain lipids without prior oxydation to acetate and resynthesis of fatty acids. Contamination by blood can be excluded. (The preparation contains all subcellular fraction except cytosol). The labelled stearic acid taken up is partly metabolized in the brain either by elongation or by degradation and in situ resynthesis of fatty acids. The activity in oleic acid and mono-unsaturated chains is hardly detectable. The labelled acids are incorporated into lipids or subcellular particles following characteristic kinetics, which show a diminution by 24 hours. When analysing the evolution of each lipid, it is shown that this profile is followed by phospholipids, but not by cerebrosides and free fatty acids. The formers are still increasing up to 50 hours, the latters are stable (suggesting a physical binding between membranes and free fatty acids). Thus nutrition is an important parameter for the synthesis of brain membranes as far as exogenous saturated fatty acids are needed.

Quantitative studies of the brain specific antigens GFA, 14-3-2, synaptin C1, D1, D2, D3 and D5 in jimpy mouse.

Seven antigens specific to the nervous tissue were measured in both Jimpy and control mice. The D5 and the GFA protein, both components of the glia, are strongly increased in the mutant while the neuronal components 14-3-2, synaptin C1, D1, D2 and D3 are unchanged.