Intraerythrocytic Plasmodium falciparum Expresses a High Affinity Facilitative Hexose Transporter
Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter ofP. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (K m = 0.48 mm) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition. Asexual stages of Plasmodium falciparum cause severe malaria and are dependent upon host glucose for energy. We have identified a glucose transporter ofP. falciparum (PfHT1) and studied its function and expression during parasite development in vitro. PfHT1 is a saturable, sodium-independent, and stereospecific transporter, which is inhibited by cytochalasin B, and has a relatively high affinity for glucose (K m = 0.48 mm) when expressed in Xenopus laevis oocytes. Competition experiments with glucose analogues show that hydroxyl groups at positions C-3 and C-4 are important for ligand binding. mRNA levels for PfHT1, assessed by the quantitative technique of tandem competitive polymerase chain reaction, are highest during the small ring stages of infection and lowest in gametocytes. Confocal immunofluorescence microscopy localizes PfHT1 to the region of the parasite plasma membrane and not to host structures. These findings have implications for development of new drug targets in malaria as well as for understanding of the pathophysiology of severe infection. When hypoglycemia complicates malaria, modeling studies suggest that the high affinity of PfHT1 is likely to increase the relative proportion of glucose taken up by parasites and thereby worsen the clinical condition. Malaria is one of the most important pathogenic protozoa and is responsible for more than 1 million deaths each year. Antimalarial drug resistance is developing rapidly in different geographical areas and is severely curtailing therapeutic options (1White N.J. J. Antimicrob. Chemother. 1992; 30: 571-585Crossref PubMed Scopus (249) Google Scholar). Asexual multiplication of intraerythrocytic Plasmodium falciparum is a prerequisite for the development of clinical symptoms and lethal outcome in malarial infection (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar). At this stage, parasites are dependent on glucose from the host as a source of energy (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar) and metabolize glucose anaerobically. Increases in metabolic demands are associated with enlargement of parasites within erythrocytes before the stage of nuclear division (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). There is an approximately 100-fold increase in the utilization of glucose by maturing parasites when compared with uninfected erythrocytes. This induced increase in uptake of glucose is accompanied by increased production and export of lactic acid by infected cells (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar).Studies on intact infected erythrocytes have suggested that P. falciparum obtains its glucose through an equilibrative mechanism (5Kirk K. Horner H.A. Kirk J. Mol. Biochem. Parasitol. 1996; 82: 195-205Crossref PubMed Scopus (79) Google Scholar) which may involve a saturable carrier associated with the parasite itself (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar). However, these types of studies assess mechanisms of glucose transport indirectly, because multiple membrane systems are involved in analysis. More detailed assessment of the enzymatic characteristics of a proposed glucose transporter from the malarial parasite can only be individually assessed in heterologous expression systems.Previously, we have hypothesized that asexual stages of parasites encode substrate-specific transporters that are located in the region of the developing parasite's plasma membrane (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar). These transporters are presumed to act in conjunction with important changes in the permeability properties of the infected erythrocyte membrane to regulate the uptake of substrates by parasites. As a preliminary test of this hypothesis, we microinjected Xenopus laevis oocytes with mRNA obtained from cultured P. falciparum and demonstrated significantly increased uptake of several substrates or analogues of metabolism including 2′-deoxy-d-glucose (2-DOG) 1The abbreviations used are: 2-DOG, 2′-deoxy-d-glucose; PfHT1, Plasmodium falciparumhexose transporter 1; GLUT1, human facilitative glucose transporter 1; 6-DOG, 6′-deoxy-d-glucose; PCR, polymerase chain reaction; TC-PCR, tandem competitive PCR; K i, half-maximal inhibition constant for carrier transport; PBS, phosphate-buffered saline 1The abbreviations used are: 2-DOG, 2′-deoxy-d-glucose; PfHT1, Plasmodium falciparumhexose transporter 1; GLUT1, human facilitative glucose transporter 1; 6-DOG, 6′-deoxy-d-glucose; PCR, polymerase chain reaction; TC-PCR, tandem competitive PCR; K i, half-maximal inhibition constant for carrier transport; PBS, phosphate-buffered saline and lactate (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar).We have now identified a parasite-encoded hexose transporter that is localized to the region of the parasite plasma membrane within the infected red cell. Quantitation of mRNA encoding this transporter during the asexual and gametocyte stages of the parasite's life cycle suggests that its expression is under developmental control. Functional studies on this transporter in Xenopus oocytes have confirmed that it is a facilitative transporter with relatively high affinity for glucose. These findings have identified a key parasite-encoded substrate transporter that is a potentially novel drug target, as well as establishing the value of a heterologous expression system for the study of malarial transport proteins.DISCUSSIONGlucose is the main energy source for asexual stages of P. falciparum (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar). The isolation of a novel parasite-encoded hexose transporter is therefore of fundamental biological interest. A number of findings suggest that PfHT1 is the major hexose transporter for intraerythrocytic parasites. We have established that PfHT1is a single copy gene with no close homologues of PfHT1discernible from examination of both Southern and Northern blots under conditions of low stringency for hybridization and washing. PfHT1 is expressed as a single transcript (Fig. 3) that varies considerably in abundance during the life cycle of P. falciparum. The pattern of expression of PfHT1 mRNA, which peaks sharply 8 h after invasion of the red cell (Fig. 4), is consistent with anticipation of a rapid rise in glucose consumption as ring forms mature to become trophozoites (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). A sustained secondary rise inPfHT1 mRNA levels may serve to maintain glucose supply as parasites begin to divide. Thus the variation in abundance of mRNA encoding PfHT1 is consistent with the changes in glucose utilization as asexual stage parasites mature in cells. Interestingly, gametocytes expressed the lowest relative abundance of PfHT1mRNA suggesting that high levels of glucose uptake may not be critical at this stage of the infection.When expressed in Xenopus oocytes, PfHT1 mediates sodium-independent uptake of hexoses (Fig. 2). The uptake of glucose analogues in intact P. falciparum-infected erythrocytes is also sodium-independent (5Kirk K. Horner H.A. Kirk J. Mol. Biochem. Parasitol. 1996; 82: 195-205Crossref PubMed Scopus (79) Google Scholar) and saturable (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar). Furthermore, the predicted K m for 6-DOG uptake into parasites (∼5 mm) derived after modeling from studies in infected erythrocytes (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar) is consistent with our K i estimate for 6-DOG (2.2 mm). Taken together these observations support assignment of PfHT1 as the principal hexose transporter in asexual stages of parasite development. A number of controversial mechanisms have been proposed to explain the increase in permeability of host-derived structures to metabolites of parasites (16Desai S. Krogstad D.J. McCleskey E.W. Nature. 1993; 362: 643-646Crossref PubMed Scopus (170) Google Scholar, 17Kirk K. Horner H. Elford B.C. Ellory J.C. Newbold C.I. J. Biol. Chem. 1994; 269: 3339-3347Abstract Full Text PDF PubMed Google Scholar, 18Lauer S.A. Rathod P.K. Ghori N. Haldar K. Science. 1997; 276: 1122-1125Crossref PubMed Scopus (196) Google Scholar, 19Pouvelle B. Spiegel R. Hsiao L. Howard R.J. Morris R.L. Thomas A.P. Taraschi T.F. Nature. 1991; 353: 73-75Crossref PubMed Scopus (184) Google Scholar). Whatever the nature of these important mechanisms, primary regulation of nutrient uptake and export of products of metabolism takes place across the parasite plasma membrane, because axenically grown parasites are capable of completing the asexual stage of development (20Trager W. Williams J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5351-5355Crossref PubMed Scopus (39) Google Scholar). Localization by confocal microscopy and Western blotting experiments demonstrate that PfHT1 is associated with the parasite plasma membrane and not the erythrocyte membrane. PfHT1 therefore provides an example of a molecular mechanism for transport of substrates into the parasite, in which substrate-specific transporters act in conjunction with alterations in surrounding membrane structures to supply nutrients to the parasite (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar).Heterologous expression systems avoid the difficulties of studying transport across the multiple membranes of the infected erythrocyte and will become increasingly important as the malaria-sequencing initiative evolves. The high AT content of parasite DNA does not interfere with expression in the Xenopus oocyte system.The isolation of a parasite-encoded facilitative hexose transporter permits studies focussing on differences between the well characterized human hexose transporter family of sequences (GLUT1 to GLUT5) and the newly isolated malarial homologue. We have already shown that PfHT1 requires an appropriately positioned hydroxyl in the C-4 of a which it from human G. Biochem. J. 1993; PubMed Scopus Google Scholar). differences may be used as a for the of to the affinity for shown by PfHT1 compared with may in important erythrocytes to when parasites mature rise to as malaria and the metabolic of hypoglycemia and lactic (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar). The enzymatic properties of PfHT1 suggest that is for infected erythrocytes to this substrate from host This may be important in because the on which is by through the R.J. The and Scholar). The of this metabolic is predicted to increase during of hypoglycemia glucose mm). These observations may also explain parasites to when glucose for for example during of hypoglycemia which of (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar) the of of a P. high affinity hexose transporter an drug because used in the of severe malaria to inhibition of glucose utilization N.J. G. Krishna S. Parasitol. 1993; PubMed Scopus Google Scholar). of PfHT1 may be rapidly as well as with in Malaria is one of the most important pathogenic protozoa and is responsible for more than 1 million deaths each year. Antimalarial drug resistance is developing rapidly in different geographical areas and is severely curtailing therapeutic options (1White N.J. J. Antimicrob. Chemother. 1992; 30: 571-585Crossref PubMed Scopus (249) Google Scholar). Asexual multiplication of intraerythrocytic Plasmodium falciparum is a prerequisite for the development of clinical symptoms and lethal outcome in malarial infection (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar). At this stage, parasites are dependent on glucose from the host as a source of energy (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar) and metabolize glucose anaerobically. Increases in metabolic demands are associated with enlargement of parasites within erythrocytes before the stage of nuclear division (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). There is an approximately 100-fold increase in the utilization of glucose by maturing parasites when compared with uninfected erythrocytes. This induced increase in uptake of glucose is accompanied by increased production and export of lactic acid by infected cells (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). on intact infected erythrocytes have suggested that P. falciparum obtains its glucose through an equilibrative mechanism (5Kirk K. Horner H.A. Kirk J. Mol. Biochem. Parasitol. 1996; 82: 195-205Crossref PubMed Scopus (79) Google Scholar) which may involve a saturable carrier associated with the parasite itself (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar). However, these types of studies assess mechanisms of glucose transport indirectly, because multiple membrane systems are involved in analysis. More detailed assessment of the enzymatic characteristics of a proposed glucose transporter from the malarial parasite can only be individually assessed in heterologous expression we have hypothesized that asexual stages of parasites encode substrate-specific transporters that are located in the region of the developing parasite's plasma membrane (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar). These transporters are presumed to act in conjunction with important changes in the permeability properties of the infected erythrocyte membrane to regulate the uptake of substrates by parasites. As a preliminary test of this hypothesis, we microinjected Xenopus laevis oocytes with mRNA obtained from cultured P. falciparum and demonstrated significantly increased uptake of several substrates or analogues of metabolism including 2′-deoxy-d-glucose (2-DOG) 1The abbreviations used are: 2-DOG, 2′-deoxy-d-glucose; PfHT1, Plasmodium falciparumhexose transporter 1; GLUT1, human facilitative glucose transporter 1; 6-DOG, 6′-deoxy-d-glucose; PCR, polymerase chain reaction; TC-PCR, tandem competitive PCR; K i, half-maximal inhibition constant for carrier transport; PBS, phosphate-buffered saline 1The abbreviations used are: 2-DOG, 2′-deoxy-d-glucose; PfHT1, Plasmodium falciparumhexose transporter 1; GLUT1, human facilitative glucose transporter 1; 6-DOG, 6′-deoxy-d-glucose; PCR, polymerase chain reaction; TC-PCR, tandem competitive PCR; K i, half-maximal inhibition constant for carrier transport; PBS, phosphate-buffered saline and lactate (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar). We have now identified a parasite-encoded hexose transporter that is localized to the region of the parasite plasma membrane within the infected red cell. Quantitation of mRNA encoding this transporter during the asexual and gametocyte stages of the parasite's life cycle suggests that its expression is under developmental control. Functional studies on this transporter in Xenopus oocytes have confirmed that it is a facilitative transporter with relatively high affinity for glucose. These findings have identified a key parasite-encoded substrate transporter that is a potentially novel drug target, as well as establishing the value of a heterologous expression system for the study of malarial transport is the main energy source for asexual stages of P. falciparum (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar). The isolation of a novel parasite-encoded hexose transporter is therefore of fundamental biological interest. A number of findings suggest that PfHT1 is the major hexose transporter for intraerythrocytic parasites. We have established that PfHT1is a single copy gene with no close homologues of PfHT1discernible from examination of both Southern and Northern blots under conditions of low stringency for hybridization and washing. PfHT1 is expressed as a single transcript (Fig. 3) that varies considerably in abundance during the life cycle of P. falciparum. The pattern of expression of PfHT1 mRNA, which peaks sharply 8 h after invasion of the red cell (Fig. 4), is consistent with anticipation of a rapid rise in glucose consumption as ring forms mature to become trophozoites (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). A sustained secondary rise inPfHT1 mRNA levels may serve to maintain glucose supply as parasites begin to divide. Thus the variation in abundance of mRNA encoding PfHT1 is consistent with the changes in glucose utilization as asexual stage parasites mature in cells. Interestingly, gametocytes expressed the lowest relative abundance of PfHT1mRNA suggesting that high levels of glucose uptake may not be critical at this stage of the infection.When expressed in Xenopus oocytes, PfHT1 mediates sodium-independent uptake of hexoses (Fig. 2). The uptake of glucose analogues in intact P. falciparum-infected erythrocytes is also sodium-independent (5Kirk K. Horner H.A. Kirk J. Mol. Biochem. Parasitol. 1996; 82: 195-205Crossref PubMed Scopus (79) Google Scholar) and saturable (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar). Furthermore, the predicted K m for 6-DOG uptake into parasites (∼5 mm) derived after modeling from studies in infected erythrocytes (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar) is consistent with our K i estimate for 6-DOG (2.2 mm). Taken together these observations support assignment of PfHT1 as the principal hexose transporter in asexual stages of parasite development. A number of controversial mechanisms have been proposed to explain the increase in permeability of host-derived structures to metabolites of parasites (16Desai S. Krogstad D.J. McCleskey E.W. Nature. 1993; 362: 643-646Crossref PubMed Scopus (170) Google Scholar, 17Kirk K. Horner H. Elford B.C. Ellory J.C. Newbold C.I. J. Biol. Chem. 1994; 269: 3339-3347Abstract Full Text PDF PubMed Google Scholar, 18Lauer S.A. Rathod P.K. Ghori N. Haldar K. Science. 1997; 276: 1122-1125Crossref PubMed Scopus (196) Google Scholar, 19Pouvelle B. Spiegel R. Hsiao L. Howard R.J. Morris R.L. Thomas A.P. Taraschi T.F. Nature. 1991; 353: 73-75Crossref PubMed Scopus (184) Google Scholar). Whatever the nature of these important mechanisms, primary regulation of nutrient uptake and export of products of metabolism takes place across the parasite plasma membrane, because axenically grown parasites are capable of completing the asexual stage of development (20Trager W. Williams J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5351-5355Crossref PubMed Scopus (39) Google Scholar). Localization by confocal microscopy and Western blotting experiments demonstrate that PfHT1 is associated with the parasite plasma membrane and not the erythrocyte membrane. PfHT1 therefore provides an example of a molecular mechanism for transport of substrates into the parasite, in which substrate-specific transporters act in conjunction with alterations in surrounding membrane structures to supply nutrients to the parasite (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar).Heterologous expression systems avoid the difficulties of studying transport across the multiple membranes of the infected erythrocyte and will become increasingly important as the malaria-sequencing initiative evolves. The high AT content of parasite DNA does not interfere with expression in the Xenopus oocyte system.The isolation of a parasite-encoded facilitative hexose transporter permits studies focussing on differences between the well characterized human hexose transporter family of sequences (GLUT1 to GLUT5) and the newly isolated malarial homologue. We have already shown that PfHT1 requires an appropriately positioned hydroxyl in the C-4 of a which it from human G. Biochem. J. 1993; PubMed Scopus Google Scholar). differences may be used as a for the of to the affinity for shown by PfHT1 compared with may in important erythrocytes to when parasites mature rise to as malaria and the metabolic of hypoglycemia and lactic (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar). The enzymatic properties of PfHT1 suggest that is for infected erythrocytes to this substrate from host This may be important in because the on which is by through the R.J. The and Scholar). The of this metabolic is predicted to increase during of hypoglycemia glucose mm). These observations may also explain parasites to when glucose for for example during of hypoglycemia which of (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar) the of of a P. high affinity hexose transporter an drug because used in the of severe malaria to inhibition of glucose utilization N.J. G. Krishna S. Parasitol. 1993; PubMed Scopus Google Scholar). of PfHT1 may be rapidly as well as with in is the main energy source for asexual stages of P. falciparum (3Sherman I.W. Microbiol. Rev. 1979; 43: 453-495Crossref PubMed Google Scholar). The isolation of a novel parasite-encoded hexose transporter is therefore of fundamental biological interest. A number of findings suggest that PfHT1 is the major hexose transporter for intraerythrocytic parasites. We have established that PfHT1is a single copy gene with no close homologues of PfHT1discernible from examination of both Southern and Northern blots under conditions of low stringency for hybridization and washing. PfHT1 is expressed as a single transcript (Fig. 3) that varies considerably in abundance during the life cycle of P. falciparum. The pattern of expression of PfHT1 mRNA, which peaks sharply 8 h after invasion of the red cell (Fig. 4), is consistent with anticipation of a rapid rise in glucose consumption as ring forms mature to become trophozoites (4Zolg J.W. Macleod A.J. Scaife J.G. Beaudoin R.L. In Vitro. 1984; 20: 205-215Crossref PubMed Scopus (49) Google Scholar). A sustained secondary rise inPfHT1 mRNA levels may serve to maintain glucose supply as parasites begin to divide. Thus the variation in abundance of mRNA encoding PfHT1 is consistent with the changes in glucose utilization as asexual stage parasites mature in cells. Interestingly, gametocytes expressed the lowest relative abundance of PfHT1mRNA suggesting that high levels of glucose uptake may not be critical at this stage of the infection. When expressed in Xenopus oocytes, PfHT1 mediates sodium-independent uptake of hexoses (Fig. 2). The uptake of glucose analogues in intact P. falciparum-infected erythrocytes is also sodium-independent (5Kirk K. Horner H.A. Kirk J. Mol. Biochem. Parasitol. 1996; 82: 195-205Crossref PubMed Scopus (79) Google Scholar) and saturable (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar). Furthermore, the predicted K m for 6-DOG uptake into parasites (∼5 mm) derived after modeling from studies in infected erythrocytes (6Goodyer I.D. Hayes D.J. Eisenthal R. Mol. Biochem. Parasitol. 1997; 84: 229-239Crossref PubMed Scopus (25) Google Scholar) is consistent with our K i estimate for 6-DOG (2.2 mm). Taken together these observations support assignment of PfHT1 as the principal hexose transporter in asexual stages of parasite development. A number of controversial mechanisms have been proposed to explain the increase in permeability of host-derived structures to metabolites of parasites (16Desai S. Krogstad D.J. McCleskey E.W. Nature. 1993; 362: 643-646Crossref PubMed Scopus (170) Google Scholar, 17Kirk K. Horner H. Elford B.C. Ellory J.C. Newbold C.I. J. Biol. Chem. 1994; 269: 3339-3347Abstract Full Text PDF PubMed Google Scholar, 18Lauer S.A. Rathod P.K. Ghori N. Haldar K. Science. 1997; 276: 1122-1125Crossref PubMed Scopus (196) Google Scholar, 19Pouvelle B. Spiegel R. Hsiao L. Howard R.J. Morris R.L. Thomas A.P. Taraschi T.F. Nature. 1991; 353: 73-75Crossref PubMed Scopus (184) Google Scholar). Whatever the nature of these important mechanisms, primary regulation of nutrient uptake and export of products of metabolism takes place across the parasite plasma membrane, because axenically grown parasites are capable of completing the asexual stage of development (20Trager W. Williams J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5351-5355Crossref PubMed Scopus (39) Google Scholar). Localization by confocal microscopy and Western blotting experiments demonstrate that PfHT1 is associated with the parasite plasma membrane and not the erythrocyte membrane. PfHT1 therefore provides an example of a molecular mechanism for transport of substrates into the parasite, in which substrate-specific transporters act in conjunction with alterations in surrounding membrane structures to supply nutrients to the parasite (7Penny J.I. Hall S.T. Woodrow C.J. Cowan G. Gero A.M. Krishna S. Mol. Biochem. Parasitol. 1998; 93: 81-89Crossref PubMed Scopus (24) Google Scholar). expression systems avoid the difficulties of studying transport across the multiple membranes of the infected erythrocyte and will become increasingly important as the malaria-sequencing initiative evolves. The high AT content of parasite DNA does not interfere with expression in the Xenopus oocyte The isolation of a parasite-encoded facilitative hexose transporter permits studies focussing on differences between the well characterized human hexose transporter family of sequences (GLUT1 to GLUT5) and the newly isolated malarial homologue. We have already shown that PfHT1 requires an appropriately positioned hydroxyl in the C-4 of a which it from human G. Biochem. J. 1993; PubMed Scopus Google Scholar). differences may be used as a for the of to the The affinity for shown by PfHT1 compared with may in important erythrocytes to when parasites mature rise to as malaria and the metabolic of hypoglycemia and lactic (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar). The enzymatic properties of PfHT1 suggest that is for infected erythrocytes to this substrate from host This may be important in because the on which is by through the R.J. The and Scholar). The of this metabolic is predicted to increase during of hypoglycemia glucose mm). These observations may also explain parasites to when glucose for for example during of hypoglycemia which of (2Newton C.R. Krishna S. Pharmacol. Ther. 1998; 79: 1-53Crossref PubMed Scopus (282) Google Scholar) the of of a P. high affinity hexose transporter an drug because used in the of severe malaria to inhibition of glucose utilization N.J. G. Krishna S. Parasitol. 1993; PubMed Scopus Google Scholar). of PfHT1 may be rapidly as well as with in We and for and for the