The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA.
Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.
Goswami
Srikanta
S
Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India.
Adhya
Samit
S
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D007895:Leishmania tropica / Q000201:enzymology*; D008081:Liposomes / Q000737:chemistry; D008969:Molecular Sequence Data; D009840:Oligomycins / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011522:Protons; D012313:RNA / Q000737:chemistry; D012343:RNA, Transfer / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D020112:Rhodamine 123 / Q000494:pharmacology
D008081:Liposomes; D009840:Oligomycins; D011522:Protons; D011994:Recombinant Proteins; D020112:Rhodamine 123; D012313:RNA; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D006180:Proton-Translocating ATPases