Transfer of two oligosaccharides to protein in a Chinese hamster ovary cell B211 which utilizes polyprenol for its N-linked glycosylation intermediates.

B211, a glycosylation mutant isolated from Chinese hamster ovary cells, synthesizes 10- to 15-fold less Glc3Man9GlcNAc2-P-P-lipid, the substrate used by the oligosaccharide transferase in the synthesis of asparagine-linked glycoproteins. B211 cells are also 10- to 15-fold deficient in the glucosylation of oligosaccharide-lipid. Despite these properties, protein glycosylation in B211 cells proceeds at a level similar to (50% of) parental cells. We asked whether the near wild-type level of glycosylation was due to the transfer of alternative oligosaccharide structures to protein in B211 cells. The aberrant size of [35S]methionine-labeled VSV G protein and the increased percentage of endoglycosidase H-resistant tryptic peptides as compared to parental cells supported this hypothesis. B211 cells were labeled with [2-3H]mannose either for 1 min or for 1 h in the presence of glycoprotein-processing inhibitors so that the oligosaccharides initially transferred to protein could be analyzed. In addition to Glc3Man9GlcNAc2, a second, endoglycosidase H-resistant oligosaccharide was transferred whose structure was determined by alpha-mannosidase digestion, gel filtration chromatography, and HPLC to be Glc0,1Man5GlcNAc2. Finally, since the synthesis of reduced amounts of Glc3Man9GlcNAc2-P-P-lipid was also a phenotype seen in another glycosylation mutant, Lec9, we analyzed the long-chain prenol in B211 cells. B211 cells synthesized and utilized polyprenol rather than dolichol for all N-linked glycosylation intermediates as determined by HPLC analysis of [3H]mevalonate-labeled lipids. Cell fusions analyzed by similar techniques indicated that B211, originally isolated as a concanavalin A-resistant cell line, is in the Lec9 complementation group.

Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene expression.

Glucose is the preferred carbon source for most eukaryotic cells and has profound effects on many cellular functions. How cells sense glucose and transduce a signal into the cell is a fundamental, unanswered question. Here we describe evidence that two unusual glucose transporters in the yeast Saccharomyces cerevisiae serve as glucose sensors that generate an intracellular glucose signal. The Snf3p high-affinity glucose transporter appears to function as a low glucose sensor, since it is required for induction of expression of several hexose transporter (HXT) genes, encoding glucose transporters, by low levels of glucose. We have identified another apparent glucose transporter, Rgt2p, that is strikingly similar to Snf3p and is required for maximal induction of gene expression in response to high levels of glucose. This suggests that Rgt2p is a high glucose-sensing counterpart to Snf3p. We identified a dominant mutation in RGT2 that causes constitutive expression of several HXT genes, even in the absence of the inducer glucose. This same mutation introduced into SNF3 also causes glucose-independent expression of HXT genes. Thus, the Rgt2p and Snf3p glucose transporters appear to act as glucose receptors that generate an intracellular glucose signal, suggesting that glucose signaling in yeast is a receptor-mediated process.

Intracellular distribution of Arf proteins in mammalian cells. Arf6 is uniquely localized to the plasma membrane.

Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells. The plasma membrane distrubution was unaffected by either GTPgammaS (guanosine 5'-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTPgammaS. The GTPgammaS-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTPgammaS or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1,2).

Ceramide as a modulator of endocytosis.

The effects of ceramide analogs on the uptake of markers for fluid-phase (horseradish peroxidase, HRP) and receptor-mediated (low density lipoprotein, LDL) endocytosis were studied in Chinese hamster fibroblasts. N-Hexanoyl-D-erythro-sphingosine (C6-Cer) decreased the uptake of HRP in a dose-dependent manner. Internalization was inhibited > 40% with 25 microM C6-Cer, relative to controls, and was apparent within 5 min. Internalization of HRP was also inhibited by other Cer analogs and by treatment with agents that raise levels of endogenous Cer (sphingomyelinase or the glycosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP)), but not by N-hexanoyl-D-erythrosphinganine (C6-dihydro-Cer) or sphingosine. Internalization of LDL was also inhibited by C6-Cer in a concentration-dependent manner, but was less pronounced than the effect on HRP internalization (10% versus 40% inhibition with 25 microM C6-Cer), suggesting that ceramide might affect fluid-phase and receptor-mediated endocytosis to different extents. C6-Cer also slowed HRP and LDL transport from endosomes to lysosomes as studied by analysis of endocytic vesicles on Percoll density gradients and induced a redistribution of endocytic organelles as determined by fluorescence microscopy of intact cells using appropriate markers. This resulted in decreased degradation of 125I-labeled LDL in the presence of C6-Cer. These results suggest that endogenous ceramide may modulate endocytosis.

ADP-ribosylation factor (ARF)-like 3, a new member of the ARF family of GTP-binding proteins cloned from human and rat tissues.

The human and rat homologues of a new member of the ADP-ribosylation factor (ARF) family of 21-kDa GTP-binding proteins, termed Arl3, were identified as an expressed sequence tag (human) and as a product of polymerase chain reaction amplification using degenerate probes derived from conserved sequences in members of the ARF family (rat). Alignments of the full-length open reading frames of the human and rat homologues revealed the encoded proteins to be over 97% identical to each other and 43% identical to human ARF1. Northern blots of mRNA from seven human tissues and four rat tissues revealed the presence of a ubiquitous band of about 1 kilobase in length that hybridized with the corresponding Arl3 probes. A number of human tumor cell lines expressed Arl3, as determined by immunoblotting with an Arl-specific antibody, raised against a peptide derived from the human Arl3 sequence. The level of Arl3 expressed in these cell lines was on the order of 0.01% of total cell protein. Purified recombinant human Arl3 was shown to bind guanine nucleotides but lacks ARF activity and intrinsic or ARF GTPase-activating protein-stimulated GTPase activity. In contrast to ARF proteins, the Arl3 protein has reduced dependence on phospholipids and magnesium for guanine nucleotide exchange. Thus, Arl3 is a ubiquitously expressed GTP-binding protein in the ARF family with distinctive biochemical properties consistent with its having unique, but unknown, role(s) in cell physiology.

Effects of the glucosphingolipid synthesis inhibitor, PDMP, on lysosomes in cultured cells.

The glucosphingolipid synthesis inhibitor, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) has a wide range of effects on cell physiology and morphology. Here, we studied the effects of high concentrations of PDMP on cells in culture and found that fluorescent analogs of PDMP targeted to the lysosomes of Chinese hamster ovary (CHO) cells. Overnight incubation of the cells in the presence of drug induced enlargement ("vacuolization") of the lysosomes. PDMP was toxic at high concentrations (> 30 microM); this finding was used to select CHO cells that exhibited increased resistance to PDMP (PDMPR cells). The PDMPR cells were approximately 2-fold more resistant to PDMP than the parental cells (CHO-P). PDMPR cells were resistant to a number of other drugs that are also lipophilic and possess a titratable amino group. The multidrug resistance exhibited by the PDMPR cells was distinct from that observed in cells (MDR cells) that overproduce the plasma membrane drug pump, P-glycoprotein. In addition, MDR cells were extremely sensitive to PDMP.

Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo.

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.

Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis.

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementation group display a distinctive profile of resistance to a variety of toxic lectins. In addition, they accumulate cis-alpha-unsaturated polyprenol and use mainly polyprenol rather than dolichol to synthesize the glycosylated lipids used in asparagine-linked glycosylation of proteins. The primary defect in these cells is thought to result from a deficiency in polyprenol reductase activity. Three new mutants were isolated and determined to have qualitatively, although not quantitatively, similar lectin resistance profiles to Lec9 cells. Two of these mutants (AbrR and RicR) also contained polyprenol rather than dolichol. The lectin resistance profile of an independent mutant which accumulates polyprenol, F2A8, was also found to be qualitatively similar to the Lec9 pattern. The relationship among these mutants was analysed in more detail by construction of cell-cell hybrids. Lectin resistance profiles of the hybrids demonstrated that AbrR, RicR and F2A8 fell into the Lec9 complementation group. Analysis of prenols in the hybrids also showed that F2A8 was a member of the Lec9 group. Surprisingly, a significant fraction of the prenols found in Lec9 x Parent hybrids was polyprenol (up to 30% of the neutral fraction), whereas the prenols found in Parent x Parent hybrids were nearly exclusively dolichol (97% of the neutral lipid fraction). Therefore, reduction of polyprenol to dolichol appears to be a rate-limiting step in the synthesis of dolichol since hybrids with differing numbers of wild-type alleles can be biochemically distinguished.

Inhibition of glycoprotein traffic through the secretory pathway by ceramide.

Incubation of vesicular stomatitis virus-infected cells with short-chain, cell-permeable ceramide (Cer) analogs decreased the rate of viral glycoprotein transport through the Golgi complex and reduced the number of infectious virions released from cells in a concentration-dependent manner. These effects appeared to be caused directly by Cer, rather than by one of its metabolites. Cer treatment also disrupted the Golgi apparatus within 1 h, although cells treated for up to 24 h with Cer remained viable. Our results suggest that endogenous Cer may modulate secretory protein traffic and that exogenously added Cer analogs may be useful as antiviral agents.

Effects of a sphingolipid synthesis inhibitor on membrane transport through the secretory pathway.

We investigated the effects of an inhibitor of sphingolipid biosynthesis, 1-phenyl-2-(decanoyl-amino)-3-morpholino-1-propanol (PDMP), on cells in culture. Two Golgi-associated enzymes were affected by incubation of cells with PDMP. The synthesis of glucosylceramide was inhibited at low concentrations of PDMP (2.5-10 microM), and in the presence of higher concentrations (greater than or equal to 25 microM), synthesis of sphingomyelin was also reduced. Transport of vesicular stomatitis virus G protein through the Golgi complex was progressively retarded by increasing concentrations of PDMP. In the presence of 75 microM PDMP, the half-times of VSV-G protein arrival at the cis, medial, and trans Golgi and the cell surface were increased 1.5-, 2.1-, 2.4-, and 2.8-fold, respectively, compared to control values. Transport of fluorescent sphingolipids, synthesized de novo at the Golgi complex from fluorescent ceramide precursors, to the cell surface was retarded by approximately 20% in the presence of 50 microM PDMP and by approximately 50% in the presence of 100 microM PDMP. Control experiments demonstrated that PDMP had minimal effects on cell morphology and physiology (including microtubule and endoplasmic reticulum structure, mitochondrial function, and endocytosis). Although incubation of cells with relatively high concentrations of PDMP was required to see the effects on protein and sphingolipid transport, use of a fluorescent analogue of PDMP demonstrated that most cell-associated PDMP was sequestered in lysosomes, while the concentration at the Golgi complex, the site of the target synthetic enzymes, was relatively low. Taken together, these results suggest that transport of proteins and sphingolipids through the secretory pathway may be coupled to sphingolipid synthesis.

Activation of 5-[125I]iodonaphthyl-1-azide via excitation of fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) lipid analogs in living cells. A potential tool for identification of compartment-specific proteins and proteins involved in intracellular transport and metabolism of lipids.

We describe a new technique for analysis of proteins located near fluorescent lipid analogs in intact living cells using the membrane-permeant, photoactivatable probe, 5-[125I]iodonaphthyl-1-azide ([125I]INA). [125I] INA can be activated directly with UV light or indirectly through excitation of adjacent fluorophores (photosensitizers) with visible light to modify nearby proteins covalently with 125I. In this report we demonstrate that fluorescent phospholipids and sphingolipids containing N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-6-aminocaproic acid serve as appropriate photosensitizers for [125I]INA. Using Chinese hamster ovary fibroblasts, we optimized the labeling conditions with respect to lipid concentration and time of irradiation and then examined the profiles of cellular proteins that were labeled when fluorescent analogs of ceramide, sphingomyelin, and phosphatidic acid were used as photosensitizers in living cells. The use of different fluorescent lipids, which label different subcellular compartments of cells as determined by fluorescence microscopy, derivatized different sets of cellular proteins with 125I. The labeled proteins were subsets of the total set of proteins available for derivatization as determined by direct activation of [125I]INA. Most proteins labeled by this procedure were pelleted by centrifugation of cell lysates at high speed (260,000 x g), but several soluble proteins were also labeled under these conditions. The implications of using this technique for identification of compartment-specific proteins and proteins involved in lipid metabolism and transport are discussed.

Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo.

We examined changes in the levels of the dolichol forms in Chinese hamster ovary cells containing alterations in the levels of activity of two enzymes in the oligosaccharyl-P-P-dolichol biosynthetic pathway, namely UDP-GlcNAc:dolichyl phosphate:GlcNAc-phosphotransferase (GlcNAc-1-phosphotransferase) and mannosylphosphoryldolichol (Man-P-Dol) synthase. Under normal conditions in wild type cells, Glc3Man9GlcNAc2-pyrophosphoryldolichol was the most abundant form. Of the other anionic forms of dolichols, dolichyl phosphate, Man-P-Dol, glucosylphosphoryldolichol, and Man5GlcNAc2-pyrophosphoryl dolichol were approximately equally abundant. When 3E11 cells (a tunicamycin-resistant Chinese hamster ovary line containing 15 times more GlcNAc-1-phosphotransferase activity than wild type), B4-2-1 cells (a mutant lacking Man-P-Dol synthase activity), and wild type cells incubated with or without tunicamycin were utilized, significant changes in the levels of most of the anionic dolichol derivatives, with the exception of dolichyl phosphate, were found. Since changes in dolichyl phosphate levels were not detected under a variety of conditions where the levels of enzyme activity utilizing this substrate were varied, all three enzymes appear to have access to the same pool of dolichyl phosphate, and further, to have similar Km values for dolichyl phosphate.

Lec9 CHO glycosylation mutants are defective in the synthesis of dolichol.

Lec9 Chinese hamster ovary cells were found previously to be defective in the synthesis of N-linked glycans. This appeared to be the result of a defect in the synthesis of oligosaccharide lipid and lipid phosphate (Rosenwald, Stanley, and Krag. 1989. Mol. Cell. Biol. 9: 914-924). In this study we analyzed the steady state levels of long-chain polyisoprenyl lipids in Lec9 cells. We found that Lec9 cells are defective in the synthesis of dolichol. They accumulated a presumed precursor to dolichol, cis-a-unsaturated polyprenol and used this lipid in the synthesis of oligosaccharide lipid. Chain lengths of the activated polyprenols in Lec9 were the same lengths as dolichols in parental cells. Lec9 cells had increased levels of monosaccharylphosphoryl lipid and decreased levels of oligosaccharylpyrophosphoryl lipid compared to parental cells. The defect in Lec9 cells was specific for dolichol synthesis, since other aspects of [3H]mevalonate metabolism in Lec9 cells were the same as in parental cells. We hypothesize that Lec9 cells are defective in polyprenol reductase activity.

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

A correlation between increased beta-1,6 branching of N-linked carbohydrates and the ability of a cell to metastasize or to form a tumor has been observed in several experimental models. Lec9 Chinese hamster ovary (CHO) mutants exhibit a drastic reduction in tumorigenicity in nude mice, and this phenotype directly correlates with their ability to attach an increased proportion of beta-1,6-branched carbohydrates to the G glycoprotein of vesicular stomatitis virus (J. Ripka, S. Shin, and P. Stanley, Mol. Cell. Biol. 6:1268-1275, 1986). In this paper we provide evidence that cellular carbohydrates from Lec9 cells also contain an increased proportion of beta-1,6-branched carbohydrates, although they do not possess significantly increased activity of the beta-1,6 branching enzyme (GlcNAc-transferase V). Biosynthetic labeling experiments show that a substantial degree of underglycosylation occurs in Lec9 cells and that this affects several classes of glycoproteins. Lec9 cells synthesize ca. 40-fold less Glc3Man9GlcNAc2-P-P-lipid and ca. 2-fold less Man5GlcNAc2-P-P-lipid than parental cells do. In addition, Lec9 cells possess ca. fivefold less protein-bound oligosaccharide intermediates, and one major species is resistant to release by endo-beta-N-acetylglucosaminidase H (endo H). Membranes of Lec9 cells exhibit normal mannosylphosphoryldolichol synthase, glucosylphosphoryldolichol synthase, and N-acetylglucosaminylphosphate transferase activities in the presence of exogenous dolichyl phosphate. However, in the absence of exogenous dolichyl phosphate, mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities are reduced in membranes of Lec9 cells, indicating that membranes of Lec9 cells are deficient in lipid phosphate. This was confirmed by analysis of lipids labeled by [3H]mevalonate, which showed that Lec9 cells have less lipid phosphate than parental CHO cells. Mechanisms by which a defect in the synthesis of dolichol-oligosaccharides might alter the degree of beta-1,6 branching in N-linked carbohydrates are discussed.

A Chinese hamster ovary cell mutant F2A8 utilizes polyprenol rather than dolichol for its lipid-dependent asparagine-linked glycosylation reactions.

Previous results suggested that F2A8, a glycosylation mutant of Chinese hamster ovary cells, had a lower amount of dolichyl phosphate available for asparagine-linked glycosylation reactions relative to parental cells. The steady-state amounts and identities of polyisoprenoid lipids were determined by incubating F2A8, its parental cell line B4-2-1, and wild-type Chinese hamster ovary cells for 24 h with [2-3H]mevalonate. The neutral lipids, ubiquinone, cholesterol, and cholesteryl esters, which were the most highly labeled from [3H]mevalonate, were labeled equally in all three cell types. In wild-type and B4-2-1 cells, mevalonate incorporation into the anionic glycosylated and phosphorylated derivatives of dolichol was 10-fold higher than into the neutral free dolichol and dolichyl esters. In contrast, in F2A8 cells, label accumulated in neutral polyisoprenol lipids, so that the ratio of neutral to anionic lipids was 1:1 rather than 1:10. In wild-type and B4-2-1 cells, the polyisoprenoid found as free alcohol and in phosphorylated and glycosylated forms was shown by high pressure liquid chromatography using a silica column to be primarily dolichol, a polyisoprenol that has a saturated terminal isoprene unit. In contrast, in F2A8 cells the polyisoprenoid found primarily as the free alcohol and in phosphorylated and glycosylated forms appeared to be completely unsaturated polyprenol. The distribution of chain lengths of the labeled polyisoprenols of F2A8, B4-2-1, and wild-type cells was the same as determined by high pressure liquid chromatography using a reverse-phase column, with the predominant chain length being 19 isoprene units. These results combined with our previous studies on the phenotype of the F2A8 mutant indicate that the unsaturated polyprenyl phosphate derivatives do not function as well as dolichyl phosphate derivatives in cellular glycosylation reactions.