ABSTRACT
RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE-containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.
Subject(s)
Algal Proteins/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Characeae/enzymology , rab GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/metabolism , Algal Proteins/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Characeae/genetics , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Phylogeny , Plant Leaves/enzymology , Plant Leaves/genetics , Point Mutation , Sequence Alignment , Sequence Homology, Amino Acid , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/geneticsABSTRACT
A microscale bioassay based on 50% inhibition of K(+), Mg(2+)-ATPase activity in a microsomal fraction isolated from Nitellopsis obtusa cells was developed. Compared to that for a plasma membrane fraction purified in a sucrose gradient, the preparation procedure for a microsomal fraction is less time consuming and the yield is substantially higher. Characteristics of the microsomal preparation proved to be similar to those of the highly purified plasma membrane preparation (Manusadzianas et al., 2002), at least for heavy metals. Sensitivity to CuSO(4) of the frozen (-8 degrees C) microsomal fraction [49 +/- 17 (SD) microM; n = 8] did not significantly differ from that of the freshly isolated one (52 +/- 30, n = 8), at least for 40 days. Toxicity of leachate water from Kairiai (northern Lithuania) solid waste landfill was assessed by taking samples from various points including temporary reservoirs and analyzing them immediately after spillage (summer 2002) and after storage for almost 2 years at 4 degrees C-6 degrees C. Two tests with the macrophytic alga Nitellopsis obtusa (Charatox, 45-min EC(50) of resting potential depolarization, and ATPase assay, IC(50) of membrane ATPase activity) and one test with the crustacean Daphnia magna (Daphtoxkit F, 48-h 50% immobilization) tests were used. In general, all three tests showed successively decreasing values of landfill leachate toxicity with an increasing degree of dilution with surface waters. The possibility of employing preserved algal preparations on demand in test batteries seems to be promising, especially in emergencies.
