Ceramide‐galactosyltransferase and Cerebroside‐sulphotransferase Localisation in Golgi Membranes Isolated by a Continuous Sucrose Gradient of Mouse Brain Microsomes

Siegrist, H P; Burkart, Thomas; Wiesmann, Ulrich; Herschkowitz, N; Spycher, M. A.

doi:10.1111/j.1471-4159.1979.tb05180.x

Cited by 57 publications

(37 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The finding that the ratio CMSP/cerebroside sulfates was identical to that of myelin suggested to us that the vesicular fraction could be constituted by "proto myelin" vesicles, a structure that was previously proposed to act as vector of some myelin components (Pasquini et al, 1987). Comparing the results obtained by us and those presented by Siegrist et al (1979), it can be observed that in the membrane fractions obtained with the continuous density gradient the activities of aryl sulfatase A, 2' ,3'-cyclic nucleotide 3'-phosphohydrolase, and 5'-nucleotidase along the gradient are distributed in a similar fashion. The distribution of thiamine pyrophosphatase, which was used in this work as marker of Golgi vesicles, differs from that of cerebroside sulfotransferase, which was used for similar purposes by Siegrist et al (1979).…”

Section: Discussionmentioning

confidence: 47%

“…The activity of succinate dehydrogenase, an enzyme used to detect mitochondria, was absent in all the tubes. Electron microscopic examination of the vesicular fraction showed that it was constituted by a population of unilamellar vesicles very similar to that described by Siegrist et al (1979) as fraction 14, probably containing lysosome-like structures. Table I shows the composition of the vesicular fraction in comparison with similar data obtained from purified myelin and from a microsomal fraction isolated from whole brain homogenate.…”

Section: Resultsmentioning

confidence: 92%

“…The total homogenate was fractionated following the procedure described by Burkart et al (1982) to obtain the post-nuclear supernatants SN, and SN,. Fraction SN,, (1 7,500g supernatant) was further subfractionated as described by Siegrist et al (1979). For this purpose, it was layered on top of a continuous sucrose density gradient ranging from 0.8 to 1.3 M sucrose with a cushion of 2 M sucrose at the bottom of the tube.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

See 2 more Smart Citations

Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane

Brown¹,

Moreno²,

Bongarzone³

et al. 1993

J of Neuroscience Research

View full text Add to dashboard Cite

The possibility that cerebroside sulfates and myelin proteolipid (PLP) could be simultaneously located in transport vesicles destined to be assembled in myelin was investigated in the brain of 20 day old rats. The brain was homogenized and fractionated according to Burkart et al. (J Biol Chem 257:3151-3156, 1982) to obtain a microsomal fraction that was further subfractionated in a linear sucrose density gradient following the procedure of Siegrist et al. (J Neurochem 33:497-504, 1979) to obtain a vesicular fraction which has been shown to transport cerebroside sulfates (Burkart et al., as above). This fraction was associated with acid hydrolase activity and had a lipid composition different from that of myelin and microsomal fractions. Studied by slab gel electrophoresis, dot blot, and Western blot analysis, using a highly specific anti-PLP antibody, it was found to contain myelin PLP. In view of previous findings of several laboratories including our own, the presence of myelin proteolipid in a vesicular fraction which is related to the transport of cerebroside sulfates gives further support to the hypothesis that the delivery of both constituents to the myelin membrane could be associated.

show abstract

Section: Discussionmentioning

confidence: 47%

Section: Resultsmentioning

confidence: 92%

Section: Subcellular Fractionationmentioning

confidence: 99%

See 1 more Smart Citation

Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane

Brown¹,

Moreno²,

Bongarzone³

et al. 1993

J of Neuroscience Research

View full text Add to dashboard Cite

show abstract

“…Because we showed here that CGalT is localized to the ER, the previously reported CGalT activity in Golgi fractions (13,21,23,45) must be accounted for by a different enzyme. We here found that the CGlcT-deficient cell mutant GM95 not only lacked the ability to synthesize GlcCer but also failed to synthesize GalCer under low UDP-Glc conditions in vitro.…”

Section: Discussionmentioning

confidence: 71%

UDP-Galactose:Ceramide Galactosyltransferase Is a Class I Integral Membrane Protein of the Endoplasmic Reticulum

Sprong¹,

Kruithof²,

Leijendekker³

et al. 1998

Journal of Biological Chemistry

176

157

View full text Add to dashboard Cite

UDP-galactose:ceramide galactosyltransferase (CGalT) transfers UDP-galactose to ceramide to form the glycosphingolipid galactosylceramide. Galactosylceramide is the major constituent of myelin and is also highly enriched in many epithelial cells, where it is thought to play an important role in lipid and protein sorting. Although the biochemical pathways of glycosphingolipid biosynthesis are relatively well understood, the localization of the enzymes involved in these processes has remained controversial. We here have raised antibodies against CGalT and shown by immunocytochemistry on ultrathin cryosections that the enzyme is localized to the endoplasmic reticulum and nuclear envelope but not to the Golgi apparatus or the plasma membrane. In pulse-chase experiments, we have observed that newly synthesized CGalT remains sensitive to endoglycosidase H, confirming the results of the morphological localization experiments. In protease protection assays, we show that the largest part of the protein, including the amino terminus, is oriented toward the lumen of the endoplasmic reticulum. CGalT enzyme activity required import of UDP-galactose into the lumen of the endoplasmic reticulum by a UDP-galactose translocator that is present in the Golgi apparatus of CHO cells but absent in CHOlec8 cells. Finally, we show that CGalT activity previously observed in Golgi membrane fractions in vitro, in the absence of UDP-glucose, is caused by UDPglucose:ceramide glucosyltransferase. Therefore all galactosylceramide synthesis occurs by CGalT in vivo in the lumen of the endoplasmic reticulum.

show abstract

“…Ceramide UDPgalactosyltransferase [CGT; 2-hydroxyacylsphingosine 1-,B-galactosyltransferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-f3-D-galactosyltransferase, EC 2.4.1.45] is the key enzyme in biosynthesis of cerebrosides (7) and catalyzes the transfer of galactose from UDPgalactose to ceramide (8). The enzyme is found in rat brain microsomes (9), Golgi-enriched fractions (10), and myelin (11). Numerous attempts have been made to isolate and purify this membrane-bound enzyme (12)(13)(14)(15).…”

mentioning

confidence: 99%

Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression.

Schulte

Stoffel

1993

Proc. Natl. Acad. Sci. U.S.A.

167

119

View full text Add to dashboard Cite

Cerebrosides and sulfatides are major glycosphingolipids of the lipid bilayer of the myelin sheath assembled by oligodendrocytes and Schwann cells during myelination. Cerebrosides are synthesized by ceramide UDPgalactosyltransferase [CGT; 2-hydroxyacylsphingosine 1-3-galactosyltransferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-13-D-galactosyltransferase, EC 2.4.1.45] with UDPgalactose and ceramide as substrates. Here we describe a purification method from microsomes of myelinating rat brains that includes ion exchange, dye ligand, and lectin affinity chromatography. The enzyme was identified as a 64-kDa high-mannose glycoprotein. A CGT-specific cDNA clone was isolated from a rat brain cDNA library using CGT oligonucleotides derived from peptide sequences. The cDNA insert encodes a polypeptide of 541 amino acid residues with a molecular weight of61,126. The polypeptide has three putative glycosylation sites and one hydrophobic domain at the C terminus. A 20-residue N-terminal signal sequence is lost during cotranslational translocation. Northern blot analysis demonstrates that CGT expression is restricted to brain tissue and is time dependent, correlating with myelin basic protein expression. In situ hybridization reveals that CGT expression is restricted to the oligodendrocyte-containing cell layers of cerebrum and cerebellum, which also express myelin basic protein. The amino acid sequence ofCGT shows significant homology to mammalian UDPglucuronyltransferases, which suggests a common evolutionary origin of these enzymes.The myelin sheath of the central nervous system (CNS) and peripheral nervous system (PNS) is a highly ordered multilayer membrane system consisting of 70-80% lipids and 20-30% proteins. During the rather short period of myelination, oligodendrocytes of the CNS and Schwann cells of the PNS synthesize and assemble these components into the myelin membrane (1, 2). In the CNS, cerebrosides and sulfatides are highly enriched in white matter (3) and contribute to the insulating properties of the myelin membrane. The main pathways for biosynthesis of these complex lipids are well known (4), and the corresponding enzymatic activities responsible for their synthesis increase rapidly during myelination and decrease markedly when myelination ceases (5, 6). Ceramide UDPgalactosyltransferase [CGT; 2-hydroxyacylsphingosine 1-,B-galactosyltransferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-f3-D-galactosyltransferase, EC 2.4.1.45] is the key enzyme in biosynthesis of cerebrosides (7) and catalyzes the transfer of galactose from UDPgalactose to ceramide (8). The enzyme is found in rat brain microsomes (9), Golgi-enriched fractions (10), and myelin (11). Numerous attempts have been made to isolate and purify this membrane-bound enzyme (12-15).Here we report purification of CGT from rat brain microsomes, its characterization at the cDNA and protein levels, and its expression in myelinating brain. t Northern blot hybridization indicates that CGT is expressed as a brain-The publication costs of t...

show abstract

Ceramide‐galactosyltransferase and Cerebroside‐sulphotransferase Localisation in Golgi Membranes Isolated by a Continuous Sucrose Gradient of Mouse Brain Microsomes

Cited by 57 publications

References 16 publications

Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane

Vesicular transport of myelin proteolipid and cerebroside sulfates to the myelin membrane

UDP-Galactose:Ceramide Galactosyltransferase Is a Class I Integral Membrane Protein of the Endoplasmic Reticulum

Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression.

Contact Info

Product

Resources

About