Proteolipid of vacuolar H(+)-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant.
Vacuolar H(+)-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast.
Yatsushiro
Shouki
S
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan. shouki@shujitsu.ac.jp
Taniguchi
Shinya
S
Mitamura
Toshihide
T
Omote
Hiroshi
H
Moriyama
Yoshinori
Y
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D017125:Genes, Protozoan / Q000235:genetics*; D005816:Genetic Complementation Test / Q000379:methods; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D017354:Point Mutation; D011510:Proteolipids / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011510:Proteolipids; D025262:Vacuolar Proton-Translocating ATPases