THE H-ATPASE FROM RETICULOCYTE ENDOSOMES RECONSTITUTED INTO LIPOSOMESACTS AS AN IRON TRANSPORTER()

The H+-ATPase from reticulocyte endosomes was purified and reconstitut ed into liposomes, and protein-dependent iron transport was observed. Reconstitution of the H+-ATPase into liposomes was performed by sonica ting a lipid mixture, with a composition similar to the reticulocyte p lasma membrane, in a buffer containing ferric citrate. The nonencapsul ated iron:citrate was removed by gel filtration and the proteoliposome s diluted into 1 mM FerroZine. Upon addition of ascorbate, an initial efflux of 2.9 +/- 0.3 x 10(-2) mu mol of iron/mg of ATPase/min and 56 +/- 7% of total internal Fe(II) was detected by formation of the Fe(II )-FerroZine complex with an absorbance at 562 nm or radioactivity of F e-59(II)-FerroZine following separation using gel filtration. Both thi osulfate and ferrocyanide could substitute for ascorbate. Citrate or E GTA could substitute for FerroZine. The initial rate of Fe(II) efflux was decreased by 41 or 17% using 100 mu M of the cation channel inhibi tor N,N'-dicyclohexylcarbodiimide or 70 mu M of the ATP hydrolysis inh ibitor N-ethylmaleimide, respectively, but was unaffected by the prese nce of ATP. The amount of iron transported was decreased 51 or 39% by 100 mu M N,N'-dicyclohexylcarbodiimide or 70 mu M of the ATPase inhibi tor 7-chloro-4-nitrobenz-2-oxa-1,3-dazole. The amount of Fe(III) trans port was 80% lower than Fe(II) when reductants were not present intern ally or externally although the apparent rate constants were identical when ascorbate was externally present. These results suggest that thi s vacuolar H+-ATPase may transport iron.