A P-GLYCOPROTEIN HOMOLOG OF PLASMODIUM-FALCIPARUM IS LOCALIZED ON THE DIGESTIVE VACUOLE P-GLYCOPROTEIN HOMOLOG OF PLASMODIUM-FALCIPARUM LOCALIZED ON THE DIGESTIVE

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pghl) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pghl is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pghl protein than other isolates which is most likely because of the increased pfmdrl gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pghl is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pghl may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein. p LASMODIUMfalciparum causes the most lethal form of human malaria and is responsible for much morbidity and a large number of deaths throughout the tropical world. One of the most important forms of control of this parasite has involved the use of antimalarial drugs, in particular, chloroquine. However, the emergence of resistance to chloroquine in the late 1950s, followed by the spread of resistance to virtually all parts of the world where malaria is endemic has severely compromised the effectiveness of this drug. The development of P. falciparum parasites that are resistant to several other drugs as well as chloroquine has created a dilemma in the choice of therapeutic and prophylactic regimes to control this disease. Chloroquine resistance in P. falciparum has striking similarities to the multi-drug resistance (MDR) ~ phenotype that has been described in mammalian tumor cells (Riordan et al., 1985; Gros et al., 1986, 1988). Multi-drug-resistant tumor cells are able to expel a large array of chemically distinct antitumor drugs thus preventing accumulation of lethal concentrations of the drug within the cell (Skovsgaard, 1978; Horio et al., 1988). This is characterized by amplification of multi-drug-resistance genes and the overexpression of a 170-kD protein termed P-glycoprotein which is an ATPbinding molecule located on the plasma membrane of the cell (see Endicott and Ling, 1989). The MDR phenotype of tumor cells can be inhibited by several drugs including calcium channel blockers. Chloroquine resistance in P. falcipa1. Abbreviations used in this paper: GST, glutathione-S-transferase; MDR, multi-drug resistant; PCR, polymerase chain reaction. rum can similarly be inhibited by many of the drugs that reverse the MDR phenotype in tumor cell lines (Martin et al., 1987; Bitonti et al., 1988). In addition, efflux of chloroquine from resistant P. falciparum parasites is 40-50-fold increased compared to chloroquine-sensitive parasites (Krogstad et al., 1987). P. falciparum has two genes (pfmdrl and pfmdr2) that are homologous to the MDR genes in mammals, and they belong to a large gene family that includes molecules such as bacterial transport proteins and the product of the recently isolated cystic fibrosis gene (CFTR) (Riordan et al., 1989). The pfmdrl gene has been found to be amplified in some chloroquine-resistant lines of P. falciparum and this can involve large segments ofDNA (Foote et al., 1989), an observation that is similar to that seen in mammalian tumor ceils (Endicott and Ling, 1989). The sequences of a large number of pfmdrl genes from both resistant and sensitive parasites have suggested that several alleles ofpfmdrl are closely linked to chloroquine resistance (Foote et al., 1990). Using 1-4 key nucleotide differences it was possible to correctly predict the chloroquine resistance or sensitivity status of 34 of 36 isolates examined. These results support the hypothesis that chloroquine resistance is multigenic, as one of the alleles linked to chloroquine resistance has a single amino acid change. If this was the only change necessary to obtain chloroquine resistance it would be expected to occur very frequently and in a multifocal pattern as is the case with pyrimethamine resistance (Cowman et al., 1988; Peterson et al., 1988). In contrast to pyrimethamine resistance, chloroquine resistance arose in © The Rockefeller University Press, 0021-9525/91/06/1033/10 $2.00 The Journal of Cell Biology, Volume 113, Number 5, June 1991 1033-1042 1033 only two parts of the worm and from these two foci, in South East Asia and South America, it has spread throughout the regions where malaria is endemic. This data is in contrast to a study that analyzed the progeny of a genetic cross between a chloroquine-resistant and -sensitive cloned line of P. falciparum which showed that chloroquine resistance segregated independently of the pfmdrl andpfmdr2 genes (Wellems et al., 1990). Sequencing of the pfmdrl gene of the chloroquine sensitive line used in the genetic cross showed that it had one of the amino acid changes linked to chloroquine resistance (Foote et al., 1990). This suggests that the chloroquine-sensitive cloned line (HB3) may have apfmdrl allele that is competent for chloroquine resistance but lacks the second gene(s) that appears to be necessary for the phenotypic expression of resistance. The protein product of the pfmdrl gene has not as yet been characterized and in this study we have analyzed the level of expression of the protein and its subcellular localization and show that it is present on the digestive vacuole of trophozoites. This localization has important implications for the normal function of the protein and its possible role in the mechanism of chloroquine resistance. Materials and Methods