Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival

Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). We found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival.     

Sigma-1 Receptor Chaperone at the ER-Mitochondrion Interface Mediates the Mitochondrion-ER-Nucleus Signaling for Cellular Survival

The membrane of the endoplasmic reticulum (ER) of a cell forms contacts directly with mitochondria whereby the contact is referred to as the mitochondrion-associated ER membrane or the MAM. Here we found that the MAM regulates cellular survival via an MAM-residing ER chaperone the sigma-1 receptor (Sig-1R) in that the Sig-1R chaperones the ER stress sensor IRE1 to facilitate inter-organelle signaling for survival. IRE1 is found in this study to be enriched at the MAM in CHO cells. We found that IRE1 is stabilized at the MAM by Sig-1Rs when cells are under ER stress. Sig-1Rs stabilize IRE1 and thus allow for conformationally correct IRE1 to dimerize into the long-lasting, activated endonuclease. The IRE1 at the MAM also responds to reactive oxygen species derived from mitochondria. Therefore, the ER-mitochondrion interface serves as an important subcellular entity in the regulation of cellular survival by enhancing the stress-responding signaling between mitochondria, ER, and nucleus.     

Sigma receptor knockdown augments dysfunction and apoptosis of beta cells induced by palmitate

Sigma-1 receptor (Sig-1R) is located in the endoplasmic reticulum (ER) and clustered on the mitochondria related endoplasmic membranes, which are involved in the regulation of nervous system disease. Here, we designed Sig-1R silence MIN6 cells and studied the influence of Sig-1R silence on beta cells. We showed Sig-1R inactivation in MIN6 cells could not only decrease cell proliferation but also inhibit cell cycle, and this inhibitory effect on cell cycle might be achieved by regulating the FoxM1/Plk1/Cenpa pathway. Moreover, Sig-1R deficiency increased MIN6 cells sensitivity to lipotoxicity, exaggerated palmitate (PA)-induced apoptosis, and impaired insulin secretion. On the other hand, ER chaperone GRP78 and ER proapoptotic molecules CHOP increased in Sig-1R knockdown MIN6 cells. The ATP level decreased and reactive oxygen species (ROS) increased in this kind of cells. Furthermore not only GRP78 and CHOP levels, but also ATP and ROS levels changed more in Sig-1R silence cells after cultured with PA. Therefore, Sig-1R deficiency exaggerated PA induced beta cells apoptosis by aggravating ER stress and mitochondrial dysfunction. Together, our study showed that Sig-1R might influence the proliferation, apoptosis, and function of beta cells.     

The UDP-Galactose:Ceramide Galactosyltransferase: Expression Pattern in Oligodendrocytes and Schwann Cells During Myelination and Substrate Preference for Hydroxyceramide

Abstract: Galactosylceramide ("galactocerebroside"; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP-galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT-expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.     

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell''s responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress.Sigma-1 receptor (Sig-1R) is expressed on endoplasmic reticulum (ER) membranes. Several functions have been attributed to Sig-1R, including regulation of ion channels such as Ca2+ and K+ channels, inhibition of Ca2+ influx through the N-methyl-D-aspartate (NMDA) receptor, modulation of the release of neurotransmitters such as dopamine, regulation of lipid distribution, cell differentiation, and behavioral sensitization to cocaine and other stimulants.¹ Recently, Sig-1R was shown to have neuroprotective activity, and several studies have demonstrated that it acts as a molecular chaperone.^{2, 3, 4} Under normal conditions, Sig-1R forms a complex with another molecular chaperone, GRP78/BiP, on the ER membrane. Under ER stress, Sig-1R dissociates from BiP, interacts with IP3 receptors, and stabilizes IP3 receptor structure.⁴Numerous studies have examined the role of Sig-1R in the pathogenesis of psychiatric diseases. Postmortem analysis has shown that Sig-1R expression is reduced in the brains of schizophrenia patients.⁵ Additionally, Sig-1R knockout mice exhibit symptoms of depression.⁶ Given these observations, it is possible that reduction of Sig-1R is a pathogenic factor in disorders such as schizophrenia and depression. Therefore, numerous synthetic compounds that bind to Sig-1R, including antidepressants and antipsychotic drugs, have been examined as therapeutic targets for these disorders.⁷ However, the results of clinical testing have not been satisfactory.⁸ One possible reason is that the effects of compounds that bind to Sig-1R cannot fully manifest because Sig-1R expression is reduced in the brains of patients with psychiatric diseases. This led us to examine whether compounds capable of inducing Sig-1R expression might be therapeutic in these diseases.When cells encounter ER stress, Sig-1R expression increases in response to activation of the PERK pathway, which is one of the cellular responses to ER stress.⁹ In addition, the induction of Sig-1R expression can repress cell death signals that accompany ER stress.⁹Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) that is widely used in clinical practice as an antidepressant. Because Flv is a potent Sig-1R agonist that exhibits a stronger affinity for Sig-1R than for other SSRIs,¹⁰ we investigated its effect on Sig-1R expression and on the cellular ER stress responses.     

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca²⁺ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP₃R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca²⁺ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP₃ receptors (InsP₃R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP₃R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP₃-induced Ca²⁺ release in hepatocytes. This can be explained by the rather low expression level expression of InsP₃R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca²⁺ signaling via an inhibitory effect on InsP₃ synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP₃ synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.     

Subtype-specific and ER lumenal environment-dependent regulation of inositol 1,4,5-trisphosphate receptor type 1 by ERp44

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular channel proteins that mediate Ca(2+) release from the endoplasmic reticulum (ER) and are involved in many biological processes and diseases. IP(3)Rs are differentially regulated by a variety of cytosolic proteins, but their regulation by ER lumenal protein(s) remains largely unexplored. In this study, we found that ERp44, an ER lumenal protein of the thioredoxin family, directly interacts with the third lumenal loop of IP(3)R type 1 (IP(3)R1) and that the interaction is dependent on pH, Ca(2+) concentration, and redox state: the presence of free cysteine residues in the loop is required. Ca(2+)-imaging experiments and single-channel recording of IP(3)R1 activity with a planar lipid bilayer system demonstrated that IP(3)R1 is directly inhibited by ERp44. Thus, ERp44 senses the environment in the ER lumen and modulates IP(3)R1 activity accordingly, which should in turn contribute to regulating both intralumenal conditions and the complex patterns of cytosolic Ca(2+) concentrations.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.     

Amatoxins,Phallotoxins, Phallolysin,and Antamanide: The Biologically Active Components of Poisonous Amanita Mushroom

Multiple mechanisms for feedback control of cholesterol synthesis converge on the rate-limiting enzyme in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase. This complex feedback regulatory system is mediated by sterol and nonsterol metabolites of mevalonate, the immediate product of reductase activity. One mechanism for feedback control of reductase involves rapid degradation of the enzyme from membranes of the endoplasmic reticulum (ER). This degradation results from the accumulation of sterols in ER membranes, which triggers binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the recruitment of a membrane-associated ubiquitin ligase called gp78 that initiates ubiquitination of reductase. Ubiquitinated reductase then becomes extracted from ER membranes and is delivered to cytosolic 26S proteasomes through an unknown mechanism that is mediated by the gp78-associated ATPase Valosin-containing protein/p97 and appears to be augmented by nonsterol isoprenoids. Here, we will highlight several advances that have led to the current view of mechanisms for sterol-accelerated, ER-associated degradation of reductase. In addition, we will discuss potential mechanisms for other aspects of the pathway such as selection of reductase for gp78-mediated ubiquitination, extraction of the ubiquitinated enzyme from ER membranes, and the contribution of Insig-mediated degradation to overall regulation of reductase in whole animals.     

CERAMIDE GLYCOSYLTRANSFERASES IN CULTURED RAT CEREBELLUM: CHANGES WITH AGE, WITH DEMYELINATION, AND WITH INHIBITION OF MYELINATION BY 5-BROMO-2''-DEOXYURIDINE OR EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS SERUM

—UDP-galactose:ceramide galactosyltransferase (CGalT) (E.C. 2.4.1.62) and UDP-glucose:ceramide glucosyltransferase (CGlcT) activities were measured in myelinating cultures of newborn rat cerebellum. Specific activities were measured at various days in vitro and the pattern of activities compared with that reported for in vivo tissue. Cultures demyelinated by incubation with media containing 22% serum from rabbits in which experimental allergic encephalomyelitis (EAE) was induced by injection with whole guinea-pig spinal cord, had 28% of CGalT specific activity and 86% of CGlcT specific activity measured in control cultures. Cultures in which myelination was inhibited by maintenance on media containing 0.15 mm -5-bromo-2′-deoxyuridine (BUdR) had 10% of CGalT specific activity and 118% of CGlcT specific activity of control cultures. Cultures in which myelination was inhibited by maintenance on media containing 2% EAE serum had 12% of the CGalT specific activity of control cultures. The data suggest that in vitro CGalT is predominantly a glial enzyme while CGlcT occurs primarily in neurons, and that the reduced CGalT activity may be involved in the mechanism of myelination inhibition by BUdR and by EAE serum.     

A novel inhibitor of ceramide trafficking from the endoplasmic reticulum to the site of sphingomyelin synthesis

Ceramide produced at the endoplasmic reticulum (ER) is transported to the lumen of the Golgi apparatus for conversion to sphingomyelin (SM). N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (HPA-12) is a novel analog of ceramide. Metabolic labeling experiments showed that HPA-12 inhibits conversion of ceramide to SM, but not to glucosylceramide, in Chinese hamster ovary cells. Cultivation of cells with HPA-12 significantly reduced the content of SM. HPA-12 did not inhibit the activity of SM synthase. The inhibition of SM formation by HPA-12 was abrogated when the Golgi apparatus was made to merge with the ER by brefeldin A. Moreover, HPA-12 inhibited redistribution of a fluorescent analog of ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-Cer), from intracellular membranes to the Golgi region. Among four stereoisomers of the drug, (1R,3R)-HPA-12, which resembles natural ceramide stereochemically, was found to be the most active, although (1R,3R)-HPA-12 did not affect ER-to-Golgi trafficking of protein. Interestingly, (1R,3R)-HPA-12 inhibited conversion of ceramide to SM little in mutant cells defective in an ATP- and cytosol-dependent pathway of ceramide transport. These results indicated that (1R,3R)-HPA-12 inhibits ceramide trafficking from the ER to the site of SM synthesis, possibly due to an antagonistic interaction with a ceramide-recognizing factor(s) involved in the ATP- and cytosol-dependent pathway.     

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABA_B receptors (GABA_BRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABA_BRs are heterodimers of GABA_B1 and GABA_B2 subunits and here we show that the trafficking and surface expression of GABA_BRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABA_BR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABA_BRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABA_B1 but decreases GABA_B2 surface expression. The increase in surface GABA_B1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABA_B2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABA_BR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.     

Glucose controls phosphoregulation of hydroxymethylglutaryl coenzyme A reductase through the protein phosphatase 2A-related phosphatase protein, Ppe1, and Insig in fission yeast

HMG-CoA reductase (HMGR) catalyzes a rate-limiting step in sterol biosynthesis and is a key control point in the feedback inhibition that regulates this pathway. Through the action of the membrane protein Insig, HMGR synthesis and degradation are regulated to maintain sterol homeostasis. The fission yeast Schizosaccharomyces pombe encodes homologs of HMGR and Insig called hmg1(+) and ins1(+), respectively. In contrast to the mammalian system, Ins1 regulates Hmg1 by a nondegradative mechanism involving phosphorylation of the Hmg1 active site. Here, we investigate the role of the Ins1-Hmg1 system in coupling glucose sensing to regulation of sterol biosynthesis. We show that Ins1-dependent Hmg1 phosphorylation is strongly induced in response to glucose withdrawal and that HMGR activity is correspondingly reduced. We also find that inability to activate Hmg1 phosphorylation under nutrient limiting conditions results in overaccumulation of sterol pathway intermediates. Furthermore, we show that regulation of Hmg1 phosphorylation requires the protein phosphatase 2A-related phosphatase Ppe1 and its regulator Sds23. These results describe a mechanism by which cells tune the rate of sterol synthesis to match nutrient availability.     

Activated inositol 1,4,5-trisphosphate receptors are modified by homogeneous Lys-48- and Lys-63-linked ubiquitin chains, but only Lys-48-linked chains are required for degradation

Inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) are large, ubiquitously expressed, endoplasmic reticulum membrane proteins that form tetrameric IP(3) and Ca(2+)-gated Ca(2+) channels. Endogenous IP(3)Rs provide very appealing tools for studying the ubiquitin-proteasome pathway in intact mammalian cells because, upon activation, they are rapidly ubiquitinated and degraded. Using mass spectrometry, we previously examined the ubiquitination of IP(3)R1 in αT3-1 pituitary gonadotrophs and found that IP(3)R1 ubiquitination is highly complex, with receptors being modified at multiple sites by monoubiquitin and polyubiquitin chains formed through both Lys-48 and Lys-63 linkages (Sliter, D. A., Kubota, K., Kirkpatrick, D. S., Alzayady, K. J., Gygi, S. P., and Wojcikiewicz, R. J. H. (2008) J. Biol. Chem. 283, 35319-35328). Here, we have extended these studies to determine whether IP(3)R2 and IP(3)R3 are similarly modified and if ubiquitination is cell type-dependent. Using mass spectrometry and linkage-specific ubiquitin antibodies, we found that all IP(3)R types are subject to ubiquitination at approximately the same locations and that, independent of cell type, IP(3)Rs are modified by monoubiquitin and Lys-48- and Lys-63-linked ubiquitin chains, although in differing proportions. Remarkably, the attached Lys-48- and Lys-63-linked ubiquitin chains are homogeneous and are segregated to separate IP(3)R subunits, and Lys-48-linked ubiquitin chains, but not Lys-63-linked chains, are required for IP(3)R degradation. Together, these data provide unique insight into the complexities of ubiquitination of an endogenous ubiquitin-proteasome pathway substrate in unperturbed mammalian cells. Importantly, although Lys-48-linked ubiquitin chains appear to trigger proteasomal degradation, the presence of Lys-63-linked ubiquitin chains suggests that ubiquitination of IP(3)Rs may have physiological consequences beyond signaling for degradation.     

Biosynthesis of Galactocerebrosides and Glucocerebrosides in Glial Cell Lines

UDP-galactose:ceramide galactosyltransferase (CGalT, EC 2.4.1.45) and UDP-glucose:ceramide glucosyltransferase (CGlcT, EC 2.4.1.80) were determined in the glial cell lines G26-20, G26-24, C6, and C6TK-. The enzymatic assay for CGalT in cultured glial cells was complicated by a rapid conversion of UDP-galactose to UDP-glucose, due to the elevated UDP-galactose-4'-epimerase activity in certain glial cell clones. It seems that mechanisms regulating UDP-galactose-4'-epimerase activity and levels of UDP sugars in the glial cell lines differ from those in brain tissue. Compared with the maximum activity of CGalT in the myelinating rat brain, the enzyme activities in the oligodendroglioma clonal cell lines G26-20 and G26-24 were 16-30 times lower. On the other hand, CGalT levels in G26-20 and G26-24 cells were comparable to the values found in young rat brain before myelination starts. No CGalT activity could be detected in C6 or C6TK- cells by the method used in this study, whereas CGlcT activity was found in all glial cell lines tested and its levels were close to the values observed in the young rat brain.     

Progressive hypoxia inhibits the de novo synthesis of galactosylceramide in cultured oligodendrocytes

Neonatal rat oligodendrocyte (OLG) cultures exposed to 6 h of gradual, progressive hypoxia in a GasPak (BBL, Becton Dickinson) apparatus were not injured or metabolically impaired, but instead showed a specific inhibition of de novo synthesis (measured by [3H]palmitic acid labeling) of the major myelin component galactosylceramide (GalCer). De novo synthesis of the 2-hydroxy fatty acid GalCer (HFA-GalCer) species, which requires O2 for its synthesis, was most severely inhibited (by 65%), while non-hydroxy GalCer species (NFA-GalCer) were less affected. The synthesis of membrane glycerophospholipids and sphingomyelin was unaffected by hypoxia. Treatment of OLG with 12 nM oligomycin, an inhibitor of mitochondrial ATP synthesis, resulted in an inhibition (by 50-60%) of synthesis of all GalCer species. [3H]Palmitate labeling of NFA-ceramide, the ungalactosylated precursor of NFA-GalCer species, increased in both hypoxia and oligomycin treatments, suggesting that the conversion of newly synthesized ceramide to GalCer was blocked. Newly synthesized HFA-ceramide did not accumulate in OLG, but the small labeled HFA-ceramide pool present during hypoxia was not converted into HFA-GalCer. Pulse-chase studies indicated that NFA- and HFA-ceramides labeled during these treatments were available for galactosylation and could be converted into GalCer upon reoxygenation. [3H]Galactose labeling of NFA-GalCer species was enhanced 2-fold in hypoxia, in contrast to the inhibition seen with [3H]palmitic acid labeling. Thus, while de novo GalCer synthesis was blocked in hypoxia, galactosylation of pre-existing ceramide pools was actually enhanced. Our evidence suggests that hypoxia results in a reversible inhibition of transport of newly synthesized ceramide from its site of synthesis to its site of galactosylation, but causes an increase in galactosylation of subcellular pools of pre-existing ceramide.