Abstract
Cadmium accumulates in the vacuole of plant cells, but the mechanism driving its transport across the vacuole membrane is not understood. Here we present evidence for Cd2+ transport via a Cd2+/H+ antiport activity into tonoplast-enriched vesicles isolated from oat roots. Experimentally, accumulation of Cd2+ into vesicles could be driven by delta pH generated by either V-type ATPase or artificially using nigericin to exchange K+ and H+ in K(+)-loaded vesicles. When tonoplast-enriched vesicles were separated on a linear sucrose gradient, NO3(-)-sensitive ATPase, total MgATPase, and delta pH-dependent Cd2+ transport equilibrated at 1.11 g/ml, the density of tonoplast membrane. Cd2+ accumulation in vesicles was accompanied by efflux of protons in a Cd2+ concentration-dependent manner characteristic of an antiport activity. The delta pH-dependent Cd2+ accumulation process showed saturation kinetics with a Km(app) of 5.5 microM. Thus the process is a candidate for transport of Cd2+ from the cytoplasm to the vacuolar sap under conditions of low as well as high Cd2+ exposure.
Highlights
Cadmium accumulates in the vacuole of plant cells, both in roots and shoots, the distribution depending on the but the mechanism driving its transport across the species
When tonoplast-enriched vesicles wereseparated on a linear sucrose gradient, NOS-sensitive ATPase, total MgATPase, and ApH-dependent Cd2+transport equilibrated at 1.11 g/ ml, the density of tonoplast membrane
In the absence of osmoticum, the ATPase activity increased 4-fold to 18 pmol of PJmg of protein/h, a rate comparable with that reported for the V-type ATPase of oats by others (Churchill and Sze, 1983;Churchill et al, 1983,1984; Wang and Sze, 1985)using similar assay conditions
Summary
(Received for publication, September 11, 1992,and in revised form, February 3, 1993). MgATP-dependent ApH generation in vesicles was measured by addition of vesicles to areaction mixture containing Buffer A with 1.5 mM Tris-ATP and 100 p M [14C]methylamine (1pCi/ml), togive a protein concentrationof 0.1-0.2 mg/ml. K+/nigericin-dependent Cd" and Ca2+uptake were monitored by addition of K+-loaded tonoplast vesicles to a K+-free reaction mixture (100-fold dilution) containing 25 mM Hepes-BTP at pH 7.0, 250 mM mannitol, and 10 p M lWCdor 4sCawith or without nigericin (5 p ~ (i)n ethanol, final ethanol concentration of 1%). The resulting membrane pellets were resuspended in 0.5 ml of Buffer A, stored overnight at -70 "C, and assayed for MgATP-dependent Cd2+transport activity after the addition of 1.5 mM Tris-ATP, 3 mM MgSO4, and 10 p M Cd2+(containing approximately0.4 pCi/ml of lWCd) asdescribed above. Pierce BCA protein assay reagent was obtained from Pierce Chemical Co., "Ca (11.1mCi/mg) was obtained from ICN Radiochemicals, Irvine, CA, and "'Cd (2.11 mCi/mg) was obtained from Du Pont-New EnglandNuclear
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