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~Fumonisins: fungal toxins that shed light on sphingolipid function Fumonisins are sphinganine analogues produced by Fusarium moniliforme and related fungi. They inhibit ceramide synthase and block the biosynthesis of complex sphingolipids, promoting accumulation Disruption of sphinganine and sphinganine of sphingolipid l-phosphate. metabolism by finmonisin B, alters cell-cell interactions, the behaviour of cell-surface proteins, the activity ofprotein kinases, the metabolism of other lipids, and cell growth and viability. This multitude of effects probably accounts for the toxicity and carcinogenicity of these mycotoxins. iVaturally occurring inhibitors of sphingolipid metabolism such as firmonisins are proving to be powerful tools for studying the diverse roles of sphingolipids in cell regulation and disease. Alfred Merrill and Dennis Liotta are in the Depts of Biochemistry and Chemistry, Emory University, Atlanta, GA 30322, USA; and Ronald Riley is at the US Dept of Agriculture, Agriculture Research Service, Toxicology and Mycotoxins Research Unit, Athens, GA 30613, USA. 218 Fumonisins are a family of mycotoxins produced by Fusarium monilifomne (Sheldon) and related fungi that are common contaminants of maize (Zea mays), sorghum and related grains throughout the worldl. The structures of these mycotoxins (Fig. 1) were first reported by W. F. 0. Marasas and co-workers in 1988 (Ref. 2), reflecting over two decades of research to explain the high incidence of oesophageal cancer in certain villages in the Transkei region of South Africal. Early in these investigations, they noticed that the villagers consume beer brewed from mouldy corn, and proceeded to identify the likely culprits as F. moniliforme and a new class of mycotoxins that they named fumonisins. These conclusions elicited widespread interest among toxicologists because F. moniliforme was also associated with two devastating and costly diseases of veterinary animals: equine leukoencephalomalacia and porcine pulmonary oedema’. Several different types of fumonisins have been characterized2 (some of which are illustrated in Fig. 1) and the total number is not known yet. The ‘B’ series includes the most prevalent species, fumonisin B, (FB,), which is both toxic and carcinogenic for animals’. Less is known about fumonisins in the ‘A series,for which the amino group has been acetylated, 0 1996 Elsevier Science PII: SOS&L8924(96)10021-O Ltd but they appear to be relatively innocuous1,3. The ‘C’ series lacks the l-methyl group and resembles another family of mycotoxins produced by AZtemaria altematu (called the AAL toxins; Fig. 1) that are responsible for stem canker disease in susceptible tomatoes4. In many parts of the world, maize is treated with alkali in the preparation of tortillas. This hydrolyses the tricarballylic acid sidechain (the ‘R’ group in Fig. 1) and might reduce the toxicitys. Many animals develop liver and kidney damage after consumption of FB,, and a few manifest severe neurotoxicity (equids) or pulmonary oedema (pigs)l. FB, also affects embryonic development6 and is hepatocarcinogenic3,7,8. FB, increases the ‘leakiness’ of the permeability barrier of endothelial cells9 and is both immunostimulatory and immunosuppressivelo. The pathogenesis of FB, poisoning, therefore, may involve widespread disruption of cellular function. It was unclear how these compounds could have such diverse effects because their structures do not have any obvious similarities to other toxins. However, we noticed that they do resemble the sphingoid base backbone of sphingolipids (cf. Figs 1 and 2), a class of membrane lipids that play many important roles in cell regulationrl. This led us to combine our knowledge of molecular toxicology and sphingolipid biochemistry to investigate whether the target of fumonisins might be sphingolipid metabolism and/or cellular functions mediated by sphingolipids. An answer was soon forthcoming: FB, completely blocked de nova sphingolipid biosynthesis by rat hepatocytes in culture at concentrations (1 PM) that could plausibly be achieved in vivo12. Inhibition of ceramide synthase FB, and other members of the ‘B’ series of fumonisins are potent, competitive inhibitors of ceramide synthase 12J3,the enzyme that catalyses the acylation of sphinganine in the de novo biosynthesis of sphingolipids and the reutilization of sphingosine derived from sphingolipid turnover (Fig. 2). Ceramide synthase inhibition has been characterized in vitro with liver and brain microsomes, as well as in intact mammalian cells in culture (e.g. hepatocytes, neurons, renal cells and macrophages inter aZia)14.FB, blocks the incorporation of radiolabelled serine into the sphingoid base backbone of (dihydro)ceramides and complex sphingolipids and prevents the conversion of sphinganine to sphingosine via addition of the 4,.5-tram double bond, which occurs after acylation of sphinganine. It also blocks the reacylation of sphingoid bases (primarily sphingosine) released by hydrolysis of more complex sphingolipids, as shown in Figure 2. As a consequence of the inhibition of ceramide synthase, FB, causes sphinganine to accumulate12 and increases the formation of sphinganine l-phosphate and cleavage of the sphingoid base backbone to fatty aldehydes and ethanolamine l-phosphate (Ref. 15; Fig. 2). This route for ethanolamine phosphate synthesis has received scant attention compared with the better known decarboxylation of phosphatidylserine. However, sphingoid base catabolism accounted for one third of the ethanolamine trends in CELL BIOLOGY (Vol. 6) June 1996 in phosphatidylethanolamine in 3774 cells treated with FB, (Ref. 15). Therefore, catabolism of sphingoid bases can make a significant contribution to the cellular ethanolamine pool. Cellular effects CH,OH C-CH,$HCOOH 0” CH,COOH of fumonisins FB, alters cell morphology16-ls, cell-cell interactions9, the behaviour of cell-surface proteins1g-21 and protein kinaseszz, the metabolism of other lipids15,23 and cell growth and viability16,ZP27. These changes are not fully understood and may have multiple causes; however, as sphingolipids are associated with each of these processes, most (or all) of the cellular effects of fumonisins are likely to be consequences of the disruption of sphingolipid metabolism. The inhibition of complex sphingolipid formation has been shown to account for the inhibition of neurite outgrowth by FB, because addition of a watersoluble ceramide analogue restored the normal phenotYPer7. Considerable attention has also been given recently to the role of sphingolipids in the targeting of proteins to the appropriate cell membrane and, especially, to the association of glycosylphosphatidylinositol (GPI)-anchored proteins with sphingolipidand cholesterol-rich domainszO. Investigations by several laboratories have shown that inhibitors of sphingolipid metabolism alter membrane polarityzl and the behaviour of GPI-anchored proteins1g,20,28, which strongly supports this association. The activities of numerous cellular protein kinases and phosphoprotein phosphatases are modulated by sphingolipidsll. Although there have been few studies of the effects of FB, on protein kinases, Jones and co-workersz2 have reported that treatment of CV-1 (African green monkey kidney) cells with FB, caused repression of protein kinase C activity and transcription dependent on the transcription factor APl, but stimulated transcription from a promoter containing a cyclic-AMP response element. Sphingoid bases are potent inhibitors of protein kinase Ci1sz9;thus, the accumulation of sphinganine may account for the repression of protein kinase C. Sphingosine and sphingosine l-phosphate have been reported to impair agonist-stimulated increases in cyclic AMP in Swiss 3T3 cells30,31and S49 lymphoma cells32,and to enhance APl activity in Swiss3T3 cells31.These findings are the opposite of the effects of FB, on CV-1 cells, but, as it is not uncommon for mediators to induce opposite responses in different cell types, these effects may be related. Effects on growth regulation The effects of sphingolipids on cell growth are complex. Free sphingoid bases both stimulate and inhibit cell growth30,31,33and, at higher concentrations, are cytotoxic34; therefore, the accumulation of sphinganine in FB,-treated cells is likely to play a role in the alteration of cell growth and viability by fumonisins. Studies with LLC-PK, cells, a renal epithelial cell line, have shown a close association between the concentrations of FB, that inhibit sphingolipid biosynthesis and cause growth inhibition and toxicity l6. In more recent studies, we have found that the toxicity is partially reversed by adding trends CH,? in CELL BIOLOGY (Vol. 6) June 1996 % Fumonisin B1 % Fumonisin B2 Fumonisin A, Fumonisin C1 NH, OH OH CH,bR CH, NH, OH OH 3 CH, OR CH, OH IYHCOCH, OH OH CH, OR CH, OH NH, OH OH Alternaria CH,OR CH, toxin “4 [R = COCH,CH(COOH)CH,COOH] FIGURE Structures 1 of the major fumonisins and related mycotoxins. P-chloroalanine to inhibit the first enzyme of this pathway (serine palmitoyltransferase) and reduce sphingoid base accumulation35. Sphingoid bases induce dephosphorylation of retinoblastoma protein (pRb), which is a tumour suppressor that can cause cell-cycle arrest in the hypophosphorylated form3”. FB, addition alone did not induce pRb dephosphorylation in MOLT-4 cells, but FB, potentiated the dephosphorylation of pRb induced by exogenous sphingosine33. Therefore, it is possible that growth inhibition by FB, could involve the promotion of pRb dephosphorylation through elevation of cellular sphingoid bases. Sphingoid bases have also been reported to induce apoptosis36, and FB, has been shown recently to induce apoptosis in cultured keratinocytes and HET-1A cells, a human oesophageal cell line immortalized with SV40 T antigen”‘. This may seem odd because the production of ceramide from the turnover of sphingomyelin is associated with apoptosis38, whereas FB, decreases the levels of ceramide and more complex sphingolipids. It appears that disruption of sphingolipid biosynthesis also triggers apoptosis, and this has been demonstrated by using another inhibitor of sphingolipid biosynthesis (ISP-l), which inhibited cell-cycle progression and induced apoptosis in CTLL-2 cells3g. By contrast, the exogenous addition of sphingoid bases stimulates DNA synthesis in growth-arrested Swiss 3T3 cells, at least in part owing to their conversion to the 1-phosphate30,40. FB, also increases [“Hlthymidine incorporation into DNA in this cell line25. There are two potential causes of growth stimulation by FB, in these cells: accumulation of sphinganine (or a sphinganine metabolite, such as sphinganine l-phosphate), or the loss of a growthinhibitory complex sphingolipid, such as a ganglioside, since several growth factor receptors are downregulated by gangliosides ll. It has been possible to 219 Sphingolipid Metabolic reactions Hexadecanal + Ethanolamine-P Serine + PalmitoyCCoA ,,,,,lne P-F (orCWanine 1 \ OH 1 -phosphate 4 f OH Sphinganine I --4 NH2 FB, Y OH OH 4 Dihydroceramide (N-acyl-sphinganine) --+ -7 NH OH OH Ceramide (N-acyl-sphingosine) K--J,+ -TAH R \/;_jljO-Headgroup Sphingomyelin and Glycolipids -TNH OH OH Ceramide (N-acyl-sphingosine) Rw R’,,,,,/NH FB, --+ OH OH Sphingosine 4 Sphinganine 1 -phosphate R-JYPo3H2 W CHO RV,J OPO,H, IJ NH, i Hexadecenal + Ethanolamine-P Bioloaical activities FB, reduced substantially the accumulation of sphingoid bases and prevented the stimulation of DNA synthesis by FB, (Ref. 24). Therefore, the growth stimulation by FB, is due to the accumulation of sphinganine rather than to a reduction in complex sphingolipids. Disruption metabolism of sphingolipid in wivo Although the effects of fumonisins on sphingolipid metabolism have been studied mostly in cell culture, sphingolipid levels in liver, kidney, lung and other organs are altered when animals are fed FB17J4.Sphingoid bases are able to traverse cell membranes because the pKa of the amino group is relatively lowzg. Therefore, as intracellular levels of sphingoid bases rise owing to inhibition of ceramide synthase, the amounts of sphinganine (and sometimes sphingosine) in serum and urine increase, and can be used as biomarkers for exposure of animals to fumonisins41. It is likely that disruption of sphingolipid metabolism is fundamental to both plant and animal diseases caused by F. moniliforme and related fungi because FB, has been shown to inhibit sphingolipid biosynthesis in Saccharomycescerevisiaez3and in plants42, including corn, the primary host for F. monilifonne (Sheldon). Possible mechanisms and carcinogenicity for the toxicity of fumonisins The toxicity of FB, in vivo may involve the accumulation of free sphingoid bases as much as (or more than) the deFIGURE 2 pletion of complex sphingolipids beA summary of the pathway of sphingolipid metabolism, the sites of inhibition of ceramide cause the symptoms appear very rapidly; synthase by fumonisin B, (FB,), and biological activities that have been associated with complex for example, ponies that consume fusphingolipids and intermediates of sphingolipid biosynthesis and turnover (and, thus, may be monisin-contaminated feed can show affected when cells are exposed to FB,). FB, inhibits sphingolipid metabolism at the sites shown. behavioural changes within days, and Therefore, it causes: accumulation of sphinganine and, sometimes, elevation of sphingosine die from equine leukoencephalomalacia owing to inhibition of the reacylation of sphingosine produced by sphingolipid turnover; in one to two weeks43. This is consistent elevation of sphingoid base 1 -phosphates; increased sphingoid base turnover to hexadecanal with the known cytotoxicity of sphin(from sphinganine 1 -phosphate), hexadecenal (from sphingosine 1 -phosphate) and goid bases for many cell types, which ethanolamine phosphate; and inhibits de nova biosynthesis of more complex sphingolipids. The has been proposed to involve the inhibiological activities of sphinganine and dihydroceramide have not been explored as extensively bition of protein kinase C (Ref. 34). Since as those of sphingosine and ceramide; however, sphinganine and sphingosine both inhibit activation of protein kinase C (for exprotein kinase C, whereas dihydroceramides are not comparable with ceramides in the activation ample, by phorbol esters) protects cells of the ceramide-activated protein phosphatase and in downstream events, such as inhibition of against apoptosis, the inhibition of progrowth and induction of apoptosis. The site of inhibition of serine palmitoyltransferase by tein kinase C might also explain why P-fluoroalanine and p-chloroalanine is also shown. Abbreviations: p-F (or Cl)alanine, sphingoid bases3(j and FB, (Ref. 37) inp-fluoroalanine or P-chloroalanine; ethanolamine-P, ethanolamine phosphate; FB,, fumonisin B,; duce apoptosis. Somewhat paradoxiCPI, glycosylphosphatidylinositol; R, CH,(CH,),,-; R’, an alkyl chain of varying length. cally, liver is strongly affected by FB, in vivo, but hepatocytes in culture are reladistinguish between these possibilities by using tively resistant to killing by FB, even though they reP-fluoroalanine to inhibit the first enzyme of de novo tain sensitivity to inhibition of sphingolipid metabolismz7. This may be due to the many changes that sphingolipid biosynthesis (Fig. 2). Incubation of Swiss 3T3 cells with P-fluoroalanine blocked overall occur when hepatocytes are placed in culture44. sphingolipid biosynthesis but did not stimulate The mechanism(s) for the carcinogenicity of fumogrowth; however, addition of P-fluoroalanine plus nisins are not known; however, FB, is not genotoxicz5 220 trends in CELL BIOLOGY (Vol. 6) June 1996 TABLE Cell 1 - ELUCIDATION OF THE FUNCTIONS OF SPHINGOLIPIDS Observations type and BY USING FUMONISIN B, conclusions Refs L Hippocampal Cultured Purkinje neurons cerebellar cells Neuroblastoma neurons, 17,20 FB, inhibited de nova sphingolipid synthesis. There was no effect on morphology of cerebellar neurons, but Purkinje cell morphology was and could be partially restored by adding a ceramide analoguea. The turnover of sphingolipids in cerebellar neurons may account for the morphological changes. 13,18 the altered slow lack of cells FB, blocked sphingosine-induced differentiation, which establishes that conversion of sphingosine to ceramide is required; FB, (and other inhibitors of sphingolipid synthesis) also diminished retinoic-acid-induced differentiation; therefore, de nova ceramide synthesis is important in differentiation. 50 CD1 4 FB, caused CD14, a glycosylphosphatidylinositol (GPI)-anchored protein, to become hypersensitive to phosphatidylinositol-specific phospholipase C. The hypersensitivity was suppressed by exogenous sphingomyelin. The results indicate that sphingolipids affect the properties of CD14 in membranes. 19 MDCK II cell clones exhibiting polarized distributions of marker proteins of the apical and basal-lateral membranes FB, caused Na+/K+-ATPase and CP-2, a GPI-anchored protein, to be delivered to both apical and basal-lateral membranes in MDCK II/C cells; addition of exogenous ceramide restored the normal apical membrane sorting. These results suggest that sphingolipids are involved in the generation of cell-surface polarity. 21 P388 and U937 FB, blocked daunorubicin-induced suggests that the biosynthesis by daunorubicin. 51 CHO (NeuroZa) FB, inhibited ganglioside synthesis and reduced axon length and branching. Addition of a ceramide analogue” restored both, which suggests that sphingolipids play a role in the formation or stabilization of axonal branches. cells transfected with cells ceramide elevation and apoptosis, which de nova of ceramide is involved in cell killing Rat hepatocytes FB, inhibited sphingolipid secretion as part of very-low-density lipoproteins (VLDL) but not the secretion of apoB, cholesterol, or phosphatidylcholine, which suggests that sphingomyelin synthesis is not required for secretion of VLDL, in contrast to the requirement for phosphatidylcholine. 44 MOLT-4 cells FB, potentiated the induction of dephosphorylation product (pRb) by sphingosine; therefore, metabolism ceramides is not necessary for them to affect pRb. 33 Xenopus laevis oocytes FB, blocked sphingosine-induced germinal-vesicle that ceramide, rather than sphingosine, is active cell cycle. Saccharomyces cerevisiae which suggests of the meiotic lipids, and the pathway, and glycerolipid 52 23 ,3-diazol-4-yl)-aminocaproyllsphingosine. and appears to behave mostly as a tumour promoter’. Many tumour promoters are mitogens, and, as discussed above, FB, stimulates DNA synthesis in growtharrested Swiss3T3 cellsz5and, therefore, can be classified as a mitogen. The cytotoxicity of FB, might also be involved since some tumour promoters are toxic for normal cells, while stimulating the proliferation of cells that have escaped the normal mechanisms of growth regulation’. in CELL BIOLOGY breakdown, in reinitiation FB, decreased growth and the synthesis of sphingolipids, neutral major phospholipids synthesized via the CDP-diacylglycerol-dependent which suggests that there is coordinate regulation of sphingolipid synthesis. a6-[N-(7-nitrobenz-2-oxa-l trends of the retinoblastoma gene of sphingoid bases to (Vol. 6) June 1996 Use of FE, to elucidate sphingolipids the cellular functions of If used properly, FB, can provide valuable information about the functions of sphingolipids. Examples of how it has been used are given in Table 1. As far as we are aware, FB, has inhibited sphingolipid biosynthesis in every eukaryotic cell type that has been tested. Therefore, it can be used to investigate whether complex sphingolipids are involved in a 221 variety of cell functions. The dose dependence and time course of inhibition vary among different cell types (as does the extent of depletion of sphingolipid mass, which is a function of the rate of synthesis versus turnover), so the conditions must be optimized for each cell type. Furthermore, because the levels of sphinganine and sphinganine l-phosphate are elevated, and these are highly bioactive compounds, care must be taken in concluding that depletion of complex sphingolipids is responsible for the observed cell changes. FB, has also proved useful in determining whether exogenously added sphingoid bases must be metabolized to ceramides to elicit their cellular effects, since this conversion can be blocked with FB, (Ref. 33). The possibility that FB, may have effects other than inhibition of sphingolipid metabolism must always be considered, and a recent report has described the inhibition of protein phosphatases45, but at much higher concentrations than are required for inhibition of ceramide synthase. As described in several of the examples in the preceding section, a definitive link between the disruption of sphingolipid metabolism and changes in cell behaviour can be established by using additional inhibitors of sphingolipid biosynthesis. That is, inhibitors of serine palmitoyltransferase (such as B-fluoroalanine or B-chloroalanine) should be able to mimic the effects of FB, if inhibition of overall sphingolipid synthesis is responsible for the downstream event, but they reverse the effects of FB, when the accumulation of free sphingoid bases (or their metabolites, such as the l-phosphates) is responsible. If the inhibition of complex sphingolipid synthesis is implicated, this can also be confirmed by addition of short-chain ceramides to bypass the inhibition of ceramide synthesis. Since FB, inhibits sphingolipid synthesis in vivo, it should be useful in establishing the roles of sphingolipids in development and other complex biological events that are not easily reproduced by cells in culture. Perspectives The discovery that fumonisins inhibit ceramide synthase has broadened the range of diseasesthat are known to involve sphingolipids to include not only genetic defects in sphingolipid hydrolases but also a wide variety of diseases caused by fungal toxins. A major challenge for sphingolipid research is to identify the ‘switch’ that determines whether sphingoid bases are growth stimulatory, growth inhibitory or cytotoxic. When found, this will most likely help explain the diverse effects of fumonisins, aswell. Just a few years ago, the only naturally occurring inhibitor of sphingolipid metabolism was cycloserine, which is a relatively non-specific inhibitor of pyridoxal5’-phosphate-dependent enzymes. But, as often happens in research, the list of microbial agents that affect sphingolipid metabolism has grown rapidly to include: fumonisins (as described in this review); the Altemaria toxins4; another family of structurally unrelated inhibitors of ceramide synthase, the australifungins, which are made by Sporonniella ausiralis46; and, several naturally occurring inhibitors of serine 222 palmitoyltransferase (lipoxamycin47, sphingofungins48, and ISP-1 or myriocin4g). Evolution has obviously given considerable attention to the development of compounds that target sphingolipid metabolism. The prevalence of such compounds in nature raises interesting questions concerning their possible roles in disease. It also presents numerous opportunities to utilize them as tools for basic research and, possibly, in the development of new classesof pharmaceutical agents. References 1 MARASAS, W. F. 0. (1994) in Foodborne Disease Handbook. Diseases caused by Viruses, Parasites, and Fungi (Vol. 2) (Hui, Y. H., Corham, 1. R., Murrell, K. D. and Cliver, D. O., eds), 2 3 4 5 6 7 8 9 10 11 pp. 521-573, Marcel Dekker BEZUIDENHOUT, C. S. et al. (1988) 1. Cbem. Sot. Cbem. Commun. 743-745 CELDERBLOM, W. C. A., KRIEK, N. P. I., MARASAS, W. F. 0. and THIEL, P. C. (1991) Carcinogenesis 12, 1247-l 251 BOTTINI, A. T., BOWEN, J. R. and GILCHRIST, D. G. (1981) Tetrahedron Lett. 22, 2723-2726 SYNDENHAM, E. W., STOCKENSTROM, S., THIEL, P. G., SHEPHARD, G. S., KOCH, K. R. and MARASAS, W. F. 0. (1995) 1. Agric. Food Chem. 43, 1198-l 201 GROSS, S. M., REDDY, R.V., ROTTINGHAUS, G. E., JOHNSON, G. and REDDY, C. S. (1994) Mycopathologia 128,ll l-l 18 RILEY, R. T., VOSS, K. A., YOO, H-S., GELDERBLOM, W. C. 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L., NIMKAR, S., JAMISON, W. C., LIOl-TA, D. C. and MERRILL, A. H., Jr (1990) Biocbim. Biophys. Acta 1051, 37-45 YOO, H-S., NORRED, W. P., SHOWKER, J. and RILEY, R. E. Toxicol. Appl. Pharmacol. (in press) OHTA, H., SWEENEY, E. A., MASAMUNE, A., YATOMI, Y., HAKOMORI, S-l. and IGARASHI, Y. (1995) Cancer Rex 55, 691-697 TOLLESON, W. H. et a/. (1996) Carcinogenesis 17, 239-249 HANNUN, Y. A. and OBEID, L. M. (1995) Trends The existence of a cell is dependent intimately on the presence of iron - too much or too little results in cell death. Cells from all tissues have an essential requirement for iron in order to proceed from Gl to S phase of the cell cycle. Iron-containing enzymes catalyse many reactions involving energy metabolism, respiration and DNA synthesis’. In general, iron metabolism is highly regulated, and iron is found complexed mainly with either specific transport proteins or storage proteins. Iron is rarely found in the blood plasma in the free state since it is highly toxic to sensitive tissues such as the liver, pancreas, heart and pituitary2. Owing to its toxicity, mechanisms have evolved to control closely the absorption and distribution of iron within the body. Intracellular iron is stored predominantly in the form of ferritin within the liver and is supplied to other tissues through the circulation of the serum iron-transport protein, transferrin (Tf). The main role of transferrin is to mop up free iron and to shuttle iron in a soluble non-toxic form among the organs and cells of the body. Although there is extensive literature concerning cellular iron uptake and regulation, the picture is still far from complete and there is considerable evidence that the process is more complex than thought previously, involving a number of defined and undefined transporters, receptor-mediated endocytosis (RME) and other uptake mechanisms. Biocbem. 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(1995) Biochem. Biophys. Res. Commun. 211, 396-403 50 RIBONI, L., PRINETTI, A., BASSI, R., CAMINITI, A. and TETTAMANTI, G. (1995) 1. Biol. Chem. 270,26868-26875 51 BOSE, R., VERHEIJ, M., HAIMOVITZ-FRIEDMAN, A., SCOTTO, K., FUKS, Z. and KOLESNICK, R. (1995) Cell 82,405414 52 STRUM, J. C., SWENSON, K. I., TURNER, J. E. and BELL, R. M. (1995) 1. Biol. Chem. 270,13541-l 3547 39 Acknowledgements We are grateful to the graduate students, postdoctoral fellows and collaborators who have been instrumental to these studies. Our research on fumonisins has been supported by funds from the NIH, the US Dept of Agriculture and the American Institute for Cancer Research. Pumping iron in the ’90s The role of iron in cell division, cell death and human disease has recently gained increased attention. iron uptake into mammalian The best studied process for cells involves transferrin and its receptor. This review discusses evidence supporting the existence of other routes by which iron can enter mammalian cells. Specifically, iron uptake by the cell-surface GPI-linked transferrin homologue, melanotransfenin orp97, is described and possible functions of this trans fen-m-independent pa thway are proposed. The predominant pathway by which cells acquire iron from Tf is by RME (Fig. 1) of diferric-Tf complexed to the transferrin receptor (TR)3. After Tf has bound free iron, it is able to bind to the TR at the cell surface. The complex is internalized into endosomes where acidification releases the iron. Tf remains bound to the TR and recycles back to the cell surface. trends 0 1996 Elsevier Science Transferrin and the uptake in CELL BIOLOGY (Vol. of iron 6) June 1996 PII: SO962-8924(96)10019-2 Ltd 223