Fig. 5.

Ca²⁺ transport mediated by BjPCR1. (A) Time-dependent Ca²⁺ uptake by protoplasts of WT and anti–BjPCR1-5 and anti–BjPCR1-17. The protoplasts were suspended in loading buffer containing 100 μM CaCl₂, 18.5 kBq of ⁴⁵CaCl₂, and 18.5 kBq of ³H₂O, and they were then incubated for the indicated periods of time. Only the intact protoplasts were collected by centrifugation. (B) Time-dependent release of Ca²⁺ from protoplasts of WT and anti-BjPCR1 plants. The protoplasts were preloaded in medium containing 100 μM CaCl₂ and 18.5 kBq of ⁴⁵CaCl₂ for 30 min, washed briefly with ice-cold bathing solution, and incubated in the bathing medium. Only intact cells were collected, and radioactive disintegrations from the samples were counted. The Ca²⁺ content was normalized against the ³H₂O content of the protoplasts. The average ± SE is shown (n = 4, N = 3). (C and D) Ca²⁺ uptake experiment in yeast microsomes isolated from Saccharomyces cerevisiae transformed with the empty vector (V) or BjPCR1 (BP1). (C) Time course of Ca²⁺ uptake by vesicles from cells transformed with V or BP1. Ca²⁺ uptake was performed in the absence (−ATP) or presence (+ATP) of 4 mM Mg-ATP in Ca transport medium containing a standard transport buffer at 25 °C for the indicated period. The microsomes were collected by filtration on a nitrocellulose filter. (D) Effects of inhibitors of ion transport on Ca²⁺ uptake by vesicles derived from BP1-expressing cells. Uptake assay was performed using yeast microsomes expressing V or BP1 in the Ca²⁺ transport medium containing 4 mM Mg-ATP (Control) plus the compounds indicated [i.e., NH₄Cl, 5 mM; vanadate, 1 mM; valinomycin, 2 μM; carbonyl cyanide m-chlorophenylhydrate (CCCP), 10 μM]. The bars represent the Ca²⁺ concentrations in vesicles expressing BjPCR1 minus those in vesicles transformed with empty vector (n = 4, N = 2). The values (%) in the graph are the rates of uptake expressed as a percentage of the control. Average values ± SE are shown (n = 3, N = 2).