Cloning and characterization of a vacuolar ATPase A subunit homologue from Plasmodium falciparum.
The distribution of the antimalarial drug chloroquine is determined to a significant extent by a transvacuolar pH gradient in Plasmodium falciparum. A proton pump similar to the vacuolar ATPase found in many cell types has been suggested to maintain a pH gradient across the membranes of acidic compartments in the parasite. In order to understand and define the components involved in the mechanism of acidification of parasite vesicles, we have cloned and characterized a gene, designated VAP-A, encoding a P. falciparum homologue of the catalytic A subunit of the vacuolar ATPase. The VAP-A gene encodes a polypeptide of 611 amino acids which shows between 56 to 61% amino acid identity over its entire length with the sequences of vacuolar ATPase A subunits from several species. The VAP-A gene exists as a single copy gene on P. falciparum chromosome 13 and gives rise to a transcript of 3.7 kb. Antibodies raised against a VAP-A gene segment expressed in Escherichia coli react specifically with a 67-kDa polypeptide, consistent with the size predicted from the sequence and with the size of the corresponding polypeptide in other organisms. The 67-kDa protein is present throughout the asexual erythrocytic cycle and is expressed at similar levels in 5 P. falciparum isolates of differing chloroquine sensitivity. Sequence analysis of the coding region of the VAP-A gene from 2 chloroquine-sensitive and 3 chloroquine-resistant isolates has shown no changes that are linked to chloroquine resistance. Therefore, a proposed chloroquine resistance-linked vacuolar acidification defect does not involve mutations in the VAP-A gene in the isolates we have studied.
Karcz
S R
SR
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia.
Herrmann
V R
VR
Cowman
A F
AF
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002738:Chloroquine / Q000494:pharmacology; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D017125:Genes, Protozoan; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D002738:Chloroquine; D000251:Adenosine Triphosphatases