Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing (86)Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb(+) or K(+) as a substrate and has a Km for Rb(+) equal to 1 mM and a Vmax equal to 31 nmol Rb(+) h(-1) 10(-6) cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K(+) to Rb(+) as a substrate; it has a Km for Rb(+) equal to 2.5 mM and a Vmax equal to 210 nmol Rb(+) h(-1) 10(-6) cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.
Expriments are reported in which the uptake of 'Rb', used as an analog of K , into cultured cels of Arabidopsis thaiana is investigated. A single transport system is found with KM = 0.34 mliolar and V.. -14 nmoles per miigram of protein per hour. This system is blocked by the metabolic inhibitor carbonyl cyanide n-chlorophenyl hydrazone (CCCP) and by cold. At high concentrations of external K' (above 1 milllar), a significant fraction of total uptake is energy-independent. No evidence is found for more than one energy-dependent uptake system or for concentration-dependent modifications of a carrier as postulated in multiphasic transport models.Rb' uptake was also examined in cultured cells derived from an "osmotic mutant" of Arabidpsis The with NaOH, and 20 g/l glucose. Suspension cultures were grown in Bellco spinner flasks in the dark at 25 C. Large aggregates of cells were removed from suspension by gravity and discarded; the cells remaining in suspension were pelleted by low speed centrifugation and washed three times with HP-7 medium lacking K+ (NH4' substituted for K+). The final suspension used for uptake studies consisted mostly of single cells with some two-and fourcell aggregates. Cells were harvested in log phase at densities between 1 x 105 and 5 x 10' cells/ml. Cell viability was estimated by microscopic inspection and a dye exclusion test using erythrocin B. Viability by these criteria was consistently about 80%Yo.Culture 105 cells/ml. One-ml aliquots of the suspension were withdrawn at appropriate intervals and the cells were collected on Millipore filters and washed three times with 3.0 ml unlabeled HP-7. The amount of isotope taken up was determined by either liquid scintillation or gas flow counting. For experiments in the presence of CCCP2 or at 4 C, there was a 30-min preincubation period before isotope was added. Seedlings were incubated completely immersed with shaking (approximately 100 oscillations/min) at 25 C in medium containing MRbCl (10 mCi/mg) at a concentration of 3 isCi/ml. They were removed from the labeling medium and placed in desorption medium (K+-free growth medium) for 10 min at 4 C. After desorption, seedlings were blotted dry, placed in scintillation vials with scintillation fluid, and the radioactivity measured. Figure 1. Cells were incubated with shaking at 25 C in HP-7 medium containing 10 ,UM KCI and 3 ,uCi/ml 'RbCl. Samples (1.0 ml) were taken at the indicated times and the radioactivity determined. Two observations are apparent: (a) both cold and the metabolic inhibitor, CCCP, at 0.01 mm, strikingly inhibit uptake; this supports the notion that transport is an active process; and (b) there is a lag of 40 to 60 min after which transport is linear. We do not understand the reason for the lag. However, we find that uptake of Rb+ into roots of intact seedlings has a similar lag, which disappears when cells are incubated in the assay medium for 2 h prior to the addition of the radioisotope (Fig. IA)
Mutant clones of Chlamydomonas reinhardtii defective for potassium transport were isolated and characterized. Of the four genes identified, three - TRK1, TRK2 and TRK3 encode high-affinity transport functions, and one gene, HKR1, encodes a low-affinity transport function. Characterization of the potassium dependence of recombinants possessing two mutant trk alleles suggests that the protein products of TRK2 and TRK3 interact functionally, and that TRK1 may serve a regulatory function. The mutant clone defective for a low-affinity potassium transporter was isolated by mutagenizing trk2-1 cells, which lack a functional high-affinity transporter, and screening surviving cells for dependence on very high potassium concentrations. The hkr1 phenotype is expressed only in the presence of trk2-1.
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