The plant inorganic pyrophosphatase does not transport K+ in vacuole membrane vesicles multilabeled with fluorescent probes for H+, K+, and membrane potential.
It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+. Proton pumping occurred in response to pyrophosphate addition in the absence of K+. No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K+.
Ros
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R
Departament de Biologia Vegetal, Facultat de Ciències Biològiques, Universitat de València, Burjassot (València), Spain.
Romieu
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C
Gibrat
R
R
Grignon
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C
D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006859:Hydrogen / Q000378:metabolism*; D043564:Inorganic Pyrophosphatase; D007425:Intracellular Membranes / Q000378:metabolism; D017136:Ion Transport; D008564:Membrane Potentials; D010944:Plants / Q000201:enzymology*; D011188:Potassium / Q000378:metabolism*; D011522:Protons; D011755:Pyrophosphatases / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism*
D005456:Fluorescent Dyes; D011522:Protons; D006859:Hydrogen; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D011188:Potassium