Inhibitory Effect Of Tunicamycin Research Articles

Tunicamycin inhibits progression of glioma cells through downregulation of the MEG-3-regulated wnt/β-catenin signaling pathway.

Glioma is derived from the oncogenic transformation of brain and spinal cord glial cells, and is one of the most common primary brain tumors. Tunicamycin (TUN) can significantly inhibit glioma growth and aggressiveness by promoting apoptosis in glioma cells. The purpose of the present study was to investigate the effects of TUN on growth of glioma cells and examine the TUN-mediated signaling pathway. The inhibitory effects of TUN on apoptosis, growth, aggressiveness and cell cycle arrest of glioma tumor cells were determined by western blotting, reverse transcription-quantitative polymerase chain reaction, apoptotic assays and immunofluorescence. The results demonstrated that treatment with TUN suppressed growth, migration and invasion of glioma carcinoma cells. In addition, TUN treatment induced apoptosis of glioma cells through downregulation of Bcl-2 and P53 expression levels. Findings also indicated that TUN suppressed proliferation and arrested the glioma cells in the S phase of the cell cycle. Further analysis of the mechanisms of TUN demonstrated that TUN treatment upregulated the expression levels of maternally expressed gene (MEG)-3, wnt and β-catenin in glioma cells. Furthermore, knockdown of MEG-3 expression reversed the TUN-decreased wnt/β-catenin signaling pathway, which subsequently also reversed the TUN-inhibited growth and aggressiveness of glioma cells. In conclusion, the findings in the present study indicated that TUN treatment inhibited growth and aggressiveness through MEG-3-mediated wnt/β-catenin signaling, suggesting that TUN may be an efficient anticancer agent for the treatment of glioma.

Tunicamycin suppresses breast cancer cell growth and metastasis via regulation of the protein kinase B/nuclear factor-κB signaling pathway.

Breast cancer is one of the most common metastatic tumor types. Reports have suggested that Tunicamycin may inhibit the aggressiveness of cancer cells by promoting their apoptosis. In the present study, the inhibitory effects of Tunicamycin were investigated and the potential molecular mechanism underlying the Tunicamycin-inhibited growth and aggressiveness of breast cancer cells was explored. In vitro assays demonstrated that Tunicamycin significantly inhibited growth and arrested the cell cycle of breast cancer cells in a dose-dependent manner, compared with control cells. Results revealed that Tunicamycin treatment suppressed the migration and invasion of breast cancer cells. Significantly increased apoptosis of breast cancer cells was observed subsequent to Tunicamycin treatment, as compared with control cells. Mechanism analysis demonstrated that Tunicamycin inhibited the protein kinase B (Akt) and nuclear factor-κB (NF-κB) signaling pathways, whilst Akt overexpression significantly cancelled out the Tunicamycin-inhibited growth and aggressiveness of breast cancer cells, as compared with control cells. In vivo assays revealed that Tunicamycin treatment significantly inhibited tumor growth and significantly prolonged the survival of tumor-bearing mice, compared with the PBS-treated group. In conclusion, these results indicate that Tunicamycin may inhibit the growth and aggressiveness of breast cancer cells via regulation of the Akt/NF-κB signaling pathway.

Tunicamycin inhibits colon carcinoma growth and aggressiveness via modulation of the ERK-JNK-mediated AKT/mTOR signaling pathway.

Epidemiology and evidence have demonstrated that colon carcinoma is one of the most common gastrointestinal tumors in the clinic. Reports have suggested that Tunicamycin significantly inhibits aggressiveness of colon carcinoma cells by promotion of apoptosis. In the present study, the inhibitory effect of tunicamycin on colon cancer cells and the potential underlying molecular mechanism was investigated. Western blotting, immunohistochemistry, apoptotic assays and immunofluorescence were used to analyze the therapeutic effects of tunicamycin on apoptosis, growth, aggressiveness and cell cycle of colon tumor cells, by downregulation of fibronectin, vimentin and E-cadherin expression levels. In vitro experiments demonstrated that tunicamycin significantly inhibited growth, migration and invasion of colon carcinoma cells. In addition, tunicamycin administration promoted apoptosis of colon carcinoma cells via upregulation of apoptotic protease activating factor 1 and cytochrome c expression levels, which are proteins that have a role in mitochondrial apoptosis signaling. Cell cycle assays revealed that tunicamycin suppressed proliferation and arrested S phase entry of colon carcinoma cells. Mechanistic analysis demonstrated that tunicamycin reduced expression and phosphorylation levels of extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and protein kinase B (AKT), and inhibited mammalian target of rapamycin (mTOR) expression levels in colon carcinoma cells. Endogenous overexpression of ERK inhibited tunicamycin-mediated downregulation of JNK, AKT and mTOR expression, which further blocked tunicamycin-mediated inhibition of growth and aggressiveness of colon carcinoma. In vivo assays revealed that tunicamycin treatment significantly inhibited tumor growth and promoted apoptosis, which led to long-term survival of tumor-bearing mice compared with the control group. In conclusion, these results suggested that tunicamycin may inhibit growth and aggressiveness of colon cancer via the ERK-JNK-mediated AKT/mTOR signaling pathway, and suggested that tunicamycin may be a potential anti-cancer agent for colon carcinoma therapy.

Tunicamycin impairs olfactory learning and synaptic plasticity in the olfactory bulb

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1μM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

Tunicamycin inhibits Toll-like receptor-activated inflammation in RAW264.7 cells by suppression of NF-κB and c-Jun activity via a mechanism that is independent of ER-stress and N-glycosylation

In this study, we investigated the effect of tunicamycin on the production of pro-inflammatory molecules in RAW264.7 macrophage cells in response to lipopolysaccharide (LPS) and Toll-like receptor (TLR) agonists. Tunicamycin caused a reduction in LPS-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). In contrast, other ER stress-inducing chemicals, such as A23187 and thapsigargin (TG), increased LPS-induced COX-2 expression and had no effect on LPS-induced iNOS, TNF-α or IL-1β expression. Furthermore, the inhibitory effect of tunicamycin on LPS-induced inflammation was not influenced by salubrinal, an ER stress inhibitor, suggesting that the anti-inflammatory effect of tunicamycin is independent of ER stress. Tunicamycin also inhibited the expression of inflammatory molecule mRNAs induced by stimulation of TLR2 (with lipoteichoic acid) or TLR3 (with polyinosinic:polycytidylic acid), which do not require myeloid differentiation protein-2 (MD2) for their activation. Moreover, inhibition of LPS-induced iNOS expression was not inhibited by castanospermine, another N-glycosylation inhibitor, suggesting that the inhibitory effect of tunicamycin on LPS-induced iNOS induction is likely independent of MD2 N-glycosylation. Tunicamycin inhibited nuclear factor-kappaB (NF-κB) activity by suppressing LPS-induced nuclear translocation of p50 and subsequent DNA binding of p50 and p65 to the NF-κB site of the iNOS promoter. Tunicamycin also inhibited the transcriptional activity of a cAMP-response element (CRE) reporter, possibly by inhibiting c-Jun activation. Therefore, we conclude that tunicamycin represses TLR-induced inflammation through suppression of NF-κB and CRE activity via a mechanism that is independent of ER-stress and N-glycosylation.

Inhibitory effects of tunicamycin and 2-deoxyglucose on thyroglobulin synthesis.

The kinetic incorporation of labelled sugars and amino acids by rat thyroid hemilobes was measured in the presence of 2-deoxyglucose and tunicamycin, inhibitors of the glycosylation of glycoproteins. With either inhibitor the carbohydrate content of exocytosed thyroglobulin was only slightly decreased (less than 20% of control) whereas the rate of exocytosis was strongly inhibited (by 60-80%). As no intracellular accumulation or proteolysis of non-glycosylated molecules was detected, the reduced rate of thyroglobulin release seems essentially due to a decrease in protein synthesis. In a whole cell system (hemilobes), it is impossible to uncouple glycosylation and protein synthesis by incubation with tunicamycin; 50 micrograms/ml tunicamycin for 270 min inhibited total [3H]-glucosamine and 14C-labelled amino acid incorporation by 65% and 33% respectively. This can be contrasted with cell-free incubation of thyroid rough microsomes where glycosylation was blocked by the same tunicamycin concentration (90% inhibition of N-[3H]acetylglucosamine transfer from UDP-N-[3H]acetylglucosamine) whilst ongoing protein synthesis was not significantly modified (less than 4% inhibition). This clearly suggests that, in thyroid follicular cells, a regulatory link exists between the synthesis of the peptide moiety of a glycoprotein and its glycosylation.

Effect of Tunicamycin on the Uptake and Incorporation of Galactose in Hymenolepis diminuta

The effects of tunicamycin (TM) on the uptake and incorporation of tritiated galactose into the tegumental membrane and carcass from adult Hymenolepis diminuta were examined to assess the potential usefulness of this inhibitor for studying the function of the tapeworm surface glycocalyx. Hymenolepis diminuta adults (11 days old) were preincubated for 1 hr, pulsed for 30 min with [3H]galactose and [14C]leucine, and chased for 2 hr; replicate experiments were conducted in which all media contained no TM or TM at 10 micrograms/ml. Tunicamycin significantly inhibited the incorporation of tritiated galactose into the tapeworm's carcass and 30,000-g tegumental membrane fraction. Incorporation of tritiated galactose into the tapeworm's tegumental surface membrane also was inhibited significantly when expressed relative to the incorporation of [14C]leucine. Tunicamycin did not affect the amounts of free, i.e., soluble, [3H]galactose or [14C]leucine recovered from the tapeworms not did it affect the short-term (2 min) uptake of [3H]galactose by tapeworms. Thus, the inhibitory effect of TM appears to be at the level of protein glycosylation rather than carbohydrate (galactose) transport. The data indicate that TM might be useful for producing tapeworm surface membranes with diminished carbohydrate moieties.

Inhibition of glycosylation decreases Na +/H + exchange activity, blocks NHE-3 transport to the membrane, and increases NHE-3 mRNA expression in LLC-PK 1 cells

Recent studies have shown that NHE-3 is the luminal Na +/H + exchanger isoform in cultured renal proximal tubule cells LLC-PK 1 and OK (J Biol Chem 1994;269:15613-8). The purpose of the current experiments was to study the role of NHE-3 glycosylation on antiporter activity in LLC-PK 1 cells. Treatment of LLC-PK 1 cells with 1.5 μg/ml tunicamycin for 24 hours, which blocks glycosylation in the endoplasmic reticulum, significantly decreased antiporter activity as assessed by acid-stimulated sodium 22 uptake (9.52 ± 1.0 nmol/mg protein in control cells vs 5.85 ± 0.7 nmol/mg protein in tunicamycin-treated cells, p < 0.01, n = 4) and sodium-dependent pH i recovery from an acid load (0.46 ± 0.05 pH/min in control cells vs 0.35 ± 0.04 pH/min in tunicamycin-treated cells, p < 0.02, n = 6). Lactate dehydrogenase (LDH) concentration in the medium was the same in both groups ( p > 0.05), indicating that the inhibitory effect of tunicamycin was not caused by cell toxicity. Northern hybridization of poly(A) + RNA from LLC-PK 1 cells illustrated that in tunicamycin-treated cells, NHE-3 mRNA expression increased threefold over control cells. Immunoblots of luminal membranes from control LLC-PK 1 cells with specific NHE-3 antiserum showed a doublet at 94 to 95 kd and a band at 90 kd. Luminal membranes from tunicamycin-treated cells showed only one strong band at 95 kd. NHE-3 immunoblots of whole cell extract from tunicamycin-treated cells showed that in addition to the 95 kd protein, an 87 kd band was also detected. These results are consistent with the possibility that the two bands in the 94 and 90 kd areas became deglycosylated and did not reach the membrane in the presence of tunicamycin. We conclude that glycosylation of the Na +/H + exchanger isoform NHE-3 is essential for antiporter activity in LLC-PK 1 cells. The results further suggest that glycosylation of NHE-3 mediates the translocation and insertion of this exchanger in the plasma membrane.

Inhibitory effects of tunicamycin on cell-cell adhesion and cell reaggregation of the cellular slime mold,Polysphondylium pallidum

To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule ofPolysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor, and examined TM's effect on cell-cell adhesion. The vegetative growth ofPolysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 μg/ml) during only the first 4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase cells ofPolysphondylium normally show EDTA-resistant (Ca2+-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time with the lengths of incubation (0 hr>4 hr >8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12 hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion ofPolysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation on glycoproteins and/or due to reduced levels of glycoproteins themselves.

Inhibitory effect of tunicamycin on the biosynthesis of N-linked oligosaccharide chains in carrots

Inhibitory effect of tunicamycin on the biosynthesis of N-linked oligosaccharide chains in carrots

Inhibition of DNA replication of adenovirus type 5 and simian virus 40 by tunicamycin

Addition of tunicamycin, a glycosylation inhibitor, to SV40-infected CV-1 cells or Ad5-infected HeLa cells at the beginning of infection was found to inhibit the accumulation of viral DNA at late times after infection. However, tunicamycin did not block viral DNA replication when added to the infected cells at late times after infection. The inhibitory effect of tunicamycin was partially reversible in the presence of acetylglucosamine, suggesting that the effect was due to glycosylation. In spite of diminished amounts of viral DNA accumulated at the late phase of infection in the presence of tunicamycin, the transcription rate of Ad5 late RNA and the amount of adenovirus late proteins and mRNA were not significantly affected by tunicamycin treatment. The inhibitory effect of tunicamycin on Ad5 DNA replication was much reduced in 293 cells which provide El a gene products in trans. Similar observation was obtained for the replication of SV40 DNA in Cos-7 cells which provide SV40 early gene products in trans. These results suggest that tunicamycin inhibits a glycosylation event induced by the early gene products of Ad5 and SV40 viruses during the early phase of infection.

The role of N-linked glycosylation in the regulation of activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and proliferation of SV40-transformed 3T3 cells

The effects of glycosylation inhibitors on the proliferation of SV40-transformed 3T3 cells (SV-3T3) were examined in vitro. Whereas swainsonine and castanospermine, which inhibit distal steps in the glycosylational processing, exerted marginal or no effects on cell proliferation, a proximal inhibitor, tunicamycin, efficiently decreased the rate of DNA synthesis and also inhibited the activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. The inhibitory effects of tunicamycin on cell proliferation could be partially reversed by addition of dolichol, a metabolite in the pathway regulated by HMG-CoA reductase. This finding suggests that tunicamycin exerts at least one of its effects on cell proliferation by modulating the activity of HMG-CoA reductase.

Étude comparée de trois souches du coronavirus de la gastroentérite transmissible: Conditions de la réplicationvirale et de la synthèse des antigènes structuraux

Purdue-115 and D-52 strains of TGEV were compared with the 188-SG strain, which was obtained by means of a survivor selection process in gastric juice of adult pig. The 188-SG strain was characterized by (a) low infectivity, (b) delayed and restricted growth associated with low and delayed RNA synthesis, and (c) a high content of structural antigens. In contrast, Purdue-115 and D-52 strains were characterized by (a) high infectivity, and (b) a normal pattern of virus replication and RNA and structural antigen synthesis. Tunicamycin induced the inhibition of synthesis of El and E2 glycoproteins (detected by the ELISA test using monoclonal and polyclonal antibodies) as well as a significant reduction in the NP protein. The inhibitory effect of tunicamycin was influenced by the cell type and virus strain.

Inhibitory effects of tunicamycin on the mixed lymphocyte reaction and mitogen-induced lymphocyte blastogenesis

The role of asparagine(Asn)-linked saccharides on the surface of lymphocytes in cellular interactions was examined by performing studies on the effects of tunicamycin (TM), which inhibits the glycosylation of proteins N-glycosylated at asparagine residues, on the mixed lymphocyte reaction (MLR) and mitogen-induced lymphocyte blastogenesis of human lymphocytes by measuring 3H-thymidine (TdR) incorporation. Responses were expressed as percentages of that of the control (MLR without TM). The lymphocyte blastogenesis in the one- and two-way MLR were, respectively, 43.1% and 48.0% of the control at 0.1 μg/ml of TM, and 5.5% and 7.2% at 1 μg/ml of TM. The inhibitory effect of TM on the one-way MLR was shown using TM-pretreated stimulator cells, TM-pretreated responder cells or both. TM blocked lymphocyte blastogenesis in the secondary as well as in the primary MLR. The inhibitory effect of TM on the two-way MLR was observed when TM was added on day 0 to day 2, but not on day 4 of incubation. TM blocked mitogen-induced lymphocyte blastogenesis by phytohemagglutinin, concanavalin A or by pokeweed mitogen. As TM had no cytotoxic effect on cultured cells, these inhibitory effects of TM were thought to be due to the loss of Asn-linked saccharides from glycoprotein of the surface of lymphocytes. These findings indicated that Asn-linked saccharides of glycoprotein on the surface of lymphocytes were important in cellular interactions that are necessary for the cellular immune response.

Effects of tunicamycin on preimplantation mouse embryos: Prevention of molecular differentiation during blastocyst formation

To examine the role of carbohydrate-containing molecules of preimplantation mouse embryos in early development, effects of tunicamycin were analyzed at the molecular level. Embryos cultured in the presence of tunicamycin (0.1 μg/ml) from earlier than the 16-cell stage did not develop into blastocysts, though cell divisions continued normally. Tunicamycin inhibited not only the synthesis of carbohydrate chains of usual N-glycosidic glycoproteins but also that of characteristic large polysaccharides ( M r > 9K) of early embryos. Expression of several polypeptides which are characteristic of the blastocyst stage (blastocyst-characteristic proteins: BCPs) was strongly inhibited in the embryos treated by tunicamycin from earlier than the 16-cell stage, while the expression was not inhibited in the embryos treated by the drug after that stage as analyzed by two-dimensional polyacrylamide gel electrophoresis. The expression of BCPs appeared to be dependent on de novo mRNA synthesis, since it was also inhibited by α-amanitin treatment. Since tunicamycin was shown not to inhibit expression of most other proteins and the bulk of mRNA, the inhibitory effects of tunicamycin appeared to be specific for the induction of BCPs. These observations suggest that the glycoprotein(s) and/or the characteristic large polysaccharides on the morula stage embryos play an essential role not only for morphological development but also for triggering differentiation at the molecular level.

The role of gp70 in the target antigen recognized by murine leukemia virus immune cytotoxic T-lymphocytes.

AbstractThe role of the murine leukemia virus (MLV) envelope glycoprotein, gp70, in the formation of the target antigen recognized by in vivo generated MLV‐immune cytotoxic T‐lymphocytes (FMR‐MLV immune T) was studied. Treatment of the Rauscher MLV‐transformed C57BL/6 target cell, RBL‐5, for 4 h with the protein glycosylation inhibitor tunicamycin, was found to render the cells resistant to lysis by FMR‐MLV immune Tc while lysis of the tunicamycin‐treated RBL‐5 cells by allogeneic H‐2b immune Tc was not decreased. This failure of FMR‐MLV‐immune Tc to lyse tunicamycintreated RBL‐5 cells correlated with an inhibitory effect of tunicamycin on the synthesis of gp70, resulting from impaired glycosylation of the gp70 precursor, gpPr90. Yet, examination of the tunicamycin‐treated RBL‐5 cell surface by lactoperoxidase 125I iodination and immunoprecipitation techniques, revealed (I) that neither unglycosylated gp70 nor gpPr90 antigens were detectable and (2) that the gp70 antigens from tunicamycin‐treated and untreated RBL‐5 cells were identical in their (I) migration pattern in SDS polyacrylamide gel electrophoresis (SDS PAGE) (2) isoelectric focusing profiles on a pH 3.5 ‐ pH 10 gradient and (3) external, lactoperoxidase accessible, 125I peptides. Further, tunicamycin‐treated RBL‐5 cells retained 33% ‐ 50% of their Rauscher MLV gp70 expression, as determined by quantitative absorption of a goat anti‐Rauscher MLV gp70 serum. When this latter result was interpreted with reference to the study by Lesley et al. (1974), who observed a linear relationship between the amount of alloantigen expressed and susceptibility to immune Tc killing, it was concluded that this quantitative change in gp70 expression could not adequately account for the failure of FMR‐MLV immune Tc to lyse tunicamycin‐treated RBL‐5 cells. It is therefore proposed that an MLV‐induced antigen other than gp70 may be recognized on RBL‐5 cells by FMR‐MLV immune Tc.

Tunicamycin inhibits the differentiation of ST 13 fibroblasts to adipocytes with suppression of the insulin binding activity

ST 13 cells are a clonal line of murine fibroblasts that are capable of differentiating into adipocyte-like cells in vitro . When the cells were maintained as a confluent monolayer, they began to accumulate lipid droplets and to exhibit a rapid increase of insulin binding activity. Tunicamycin, a specific inhibitor of dolichol-mediated protein glycosylation, blocked this adipose conversion without affecting cell growth and total protein synthesis. The inhibitory effect of tunicamycin was dose-dependent and reversible. Enhancement of the incorporation of [ 14C]acetate into triglyceride fraction accompanying the adipose conversion was completely inhibited by tunicamycin, whereas the incorporation into phospholipid fraction was only partially affected. The insulin binding activity increased about 10-fold during differentiation, but was completely suppressed in tunicamycin-treated cells.

Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.

Tunicamycin inhibition of [ 3H]glucosamine incorporation into yeast glycoproteins: Binding of tunicamycin and interaction with phospholipids

Protoplasts of Saccharomyces strain 1016 took up [ 3H]glucosamine in the presence of an energy source; mannose was chosen to minimize randomization. It accumulated in the soluble intracellular pool primarily as UDP- N-acetyl[ 3H]glucosamine along with a small amount of [ 3H]glucosamine 6-phosphate. The antibiotic tunicamycin (TM) at 10 μg/ml did not affect the levels of these metabolites or inhibit the formation of the Nacetylglucosamine polymer, chitin, but did prevent the incorporation of [ 3H]glucosamine into mannan peptides and the synthesis of invertase. In vitro incorporation of [ 14C]mannose from GDP-[ 14C]mannose into mannan in a membrane preparation was not sensitive to 100 μg of TM/ml. TM appears to inhibit an N-acetylglucosaminyl transferase essential for glycoprotein biosynthesis. Binding of [ 3H]TM reflects its association with the plasma membrane fraction. This material could be recovered in an unaltered form by extraction with chloroform/methanol. If 0.2% phosphatidyl choline or phosphatidyl serine was added simultaneously with the [ 3H]TM, the binding of [ 3H]TM was greatly reduced, and the inhibitory effects of TM on protoplasts were prevented; however, addition of phospholipid 20 min later did not eliminate the inhibition, although about 80% of the bound [ 3H]TM was removed. TM interacts with lipophilic membrane components as well as inhibiting glycoprotein synthesis.