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Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A {3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one}-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport. —Mohammadi, A., R. J. Perry, M. K. Storey, H. W. Cook, D. M. Byers, and N. D. Ridgway. Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells. J. Lipid Res. 2001. 42: 1062–1071.

A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium. This growth defect is reversed by either ethanolamine or choline supplementation. Incubation of mutant cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells. Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain. Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of [(3)H]inositol into both proteins and lipids. Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant. We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells. These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis.

Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4-6-fold increase in [(3)H]choline incorporation into PtdCho and 10-15-fold increase in intracellular [(3)H]glycerophosphocholine. [(3)H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase alpha (CCTalpha). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [(3)H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [(3)H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTalpha protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTalpha in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTalpha to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTalpha by fatty acids or a fatty acid-derived signal.

Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30–50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents {3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone}, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.

Phosphatidylcholine (PtdCho) biosynthesis was examined in the sterol regulatory defective (SRD) Chinese hamster ovary (CHO) cell line SRD 6. SRD 6 cells do not display transcriptional activation of sterol-regulated genes and are cholesterol auxotrophs. Compared to CHO 7 cells (parental line from which the SRD cells were derived), incorporation of [3H]choline during a 2-h pulse into PtdCho and sphingomyelin was reduced 3- and 4.5-fold, respectively, in SRD 6 cells grown with or without cholesterol. SRD 6 cells grown in cholesterol-free medium for 24 h had 8% less phosphatidylcholine (PtdCho) mass compared to CHO 7 cells. Consistent with impaired CTP:phosphocholine cytidylyltransferase activity, [3H]choline-labeled SRD 6 cells had elevated [3H]phosphocholine and delayed conversion to [3H]PtdCho during a 2-h chase period. Compared to CHO 7 cells, cytosolic cytidylyltransferase activity was elevated 2- to 3-fold in SRD 6 cells grown in the absence of cholesterol, but activity in the total membrane fraction was normal. Immunoblot analysis confirmed that cytidylyltransferase mass was increased 2-fold in SRD 6 total cell extracts and cytosol, but not membranes. The amount of [32P]phosphate-labeled cytidylyltransferase in cytosol and membranes of SRD 6 cells were similar to controls. Likewise, cytidylyltransferase mRNA levels were not significantly different between SRD 6 and CHO 7. The defect in PtdCho synthesis in SRD 6 cells could be overcome by treatment with 150 microns oleate, such that after 5 h [3H] choline incorporation into PtdCho and phosphocholine in SRD 6 and CHO 7 cells was similar. Cholesterol auxotrophic SRD 6 cells display reduced PtdCho mass and synthesis and elevated levels of cytosolic cytidylyltransferase, defects that were only partially corrected by growth in exogenous cholesterol. These results indicate a requirement for normal cholesterol regulation and synthesis in the maintenance PtdCho levels and activity of cytidylyltransferase.

The lethal consequences of imbalances in lipid and sterol metabolism in human diseases such as atherosclerosis and lipid storage disorders underscores our need to know how cholesterol, phospholipid and sphingolipid metabolism is integrated. Accumulation and abnormal localization of lipids and sterol affects cellular function not only by perturbing membrane activity but also by increasing production of bioactive lipids derived from cholesterol, phospholipids and sphingolipids. For example in the NPC mouse model, accumulation of intracellular cholesterol and sphingomyelin is accompanied by increased sphingosine [187], a potent regular of protein kinase C and cell proliferation [152]. Oxidized LDL has an important role in the pathology of atherosclerosis by promoting foam cell formation and cytotoxicity [65]. 7-Hydroxycholesterol and 7-ketocholesterol are involved in many aspects of oxidized LDL activity including initiation of apoptosis in a number of cell types [188, 189] and enhancing cholesterol accumulation by inhibiting efflux [190]. Oxysterols formed intracellularly or from oxidized lipoproteins could have an important role in regulating lipid metabolism in the foam cell. Bioactive metabolites of phospholipids, such as diglyceride, phosphatidic acid and lysolipids, could also increase in circumstances of elevated deposition and have profound and varied effects on cell physiology. In addition to elucidating mechanisms for integration of lipid metabolism, we should determine when these responses go awry and assess the influence of bioactive compounds formed under these circumstances on cell viability and growth.

Ethanolamine kinase (ATP:ethanolamine O-phosphotransferase, EC 2.7.1. 82) catalyzes the committed step of phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. The gene encoding ethanolamine kinase (EKI1) was identified from the Saccharomyces Genome Data Base (locus YDR147W) based on its homology to the Saccharomyces cerevisiae CKI1-encoded choline kinase, which also exhibits ethanolamine kinase activity. The EKI1 gene was isolated and used to construct eki1Delta and eki1Delta cki1Delta mutants. A multicopy plasmid containing the EKI1 gene directed the overexpression of ethanolamine kinase activity in wild-type, eki1Delta mutant, cki1Delta mutant, and eki1Delta cki1Delta double mutant cells. The heterologous expression of the S. cerevisiae EKI1 gene in Sf-9 insect cells resulted in a 165,500-fold overexpression of ethanolamine kinase activity relative to control insect cells. The EKI1 gene product also exhibited choline kinase activity. Biochemical analyses of the enzyme expressed in insect cells, in eki1Delta mutants, and in cki1Delta mutants indicated that ethanolamine was the preferred substrate. The eki1Delta mutant did not exhibit a growth phenotype. Biochemical analyses of eki1Delta, cki1Delta, and eki1Delta cki1Delta mutants showed that the EKI1 and CKI1 gene products encoded all of the ethanolamine kinase and choline kinase activities in S. cerevisiae. In vivo labeling experiments showed that the EKI1 and CKI1 gene products had overlapping functions with respect to phospholipid synthesis. Whereas the EKI1 gene product was primarily responsible for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway, the CKI1 gene product was primarily responsible for phosphatidylcholine synthesis via the CDP-choline pathway. Unlike cki1Delta mutants, eki1Delta mutants did not suppress the essential function of Sec14p.

Two yeast enzymes, Psd1p and Psd2p, catalyze the decarboxylation of phosphatidylserine to produce phosphatidylethanolamine (PtdEtn). Mitochondrial Psd1p provides approximately 90% of total cellular phosphatidylserine decarboxylase activity. When the PSD1 gene is deleted, the resultant strain (psd1Delta) grows normally at 30 degrees C in glucose and in the absence of exogenous choline or ethanolamine. However, at elevated temperature (37 degrees C) or on the nonfermentable carbon source lactate, the growth of psd1Delta strains is minimal without ethanolamine supplementation. The reduced growth and viability correlate with a PtdEtn content below 4% of total phospholipid. These results suggest that there is a critical level of PtdEtn required to support growth. This theory is supported by growth data revealing that a psd1Delta psd2Delta dpl1Delta strain can only grow in the presence of ethanolamine. In contrast, a psd1Delta psd2Delta strain, which makes low levels of PtdEtn from sphingolipid breakdown, can be rescued by ethanolamine, choline, or the ethanolamine analogue propanolamine. psd1Delta psd2Delta cells grown in 2 mm propanolamine accumulate a novel lipid, which was determined by mass spectrometry to be phosphatidylpropanolamine (PtdPrn). PtdPrn can comprise up to 40% of the total phospholipid content in supplemented cells at the expense of phosphatidylcholine and PtdEtn. The absolute level of PtdEtn required for growth when PtdPrn is present appears to be 1% of the total phospholipid content. The essential function of the PtdEtn in the presence of propanolamine does not appear to be the formation of hexagonal phase lipid, insofar as PtdPrn readily forms hexagonal phase structures detectable by (31)P NMR.