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The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

ABSTRACT: The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.

SUBMITTER: Hayashi T

PROVIDER: S-EPMC3522309 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

Hayashi Teruo T Hayashi Eri E Fujimoto Michiko M Sprong Hein H Su Tsung-Ping TP

The Journal of biological chemistry 20121026 51

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylat ...[more]

PMID: 23105111

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Project description:The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.

Dataset Information

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

Publications

The lifetime of UDP-galactose:ceramide galactosyltransferase is controlled by a distinct endoplasmic reticulum-associated degradation (ERAD) regulated by sigma-1 receptor chaperones.

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