Immunological and functional localization of both F-type and P-type ATPases in cyanobacterial plasma membranes

Plasma and thylakoid membranes were prepared and purified from cyanobacteria Anacystis nidulans (Synechococcus PCC6301), Synechocystis PCC6803, and Anabaena PCC7120 grown photoautotrophically in axenic batch cultures and harvested either during early exponential growth (< 1 microliter packed cell mass/ml medium; light-saturated cells) or during linear growth (2-3 microliters packed cell mass/ml medium; light-limited cells). ATPase activities of methanol-activated membranes were determined by measuring (a) total inorganic phosphate released from added ATP, or (b) 32P(i) released from added [gamma-32P] ATP, or (c) NADH oxidation in a coupled ATPase/pyruvate kinase/lactate dehydrogenase system. In addition, membrane proteins were immunoblotted with antibodies raised against chloroplast and mitochondrial F1 coupling factor beta subunits, and Arabidopsis plasma membrane (P-type) ATPase, respectively. The results indicate the presence of (constitutive) P-type ATPase, inhibited by dicyclohexyl carbodiimide (DCCD), o-vanadate and diethylstilbestrol (DES) but insensitive to 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD), and of (inducible) F-type ATPase, inhibited by DCCD and NBD but insensitive to o-vanadate and DES, in the plasma membrane. Thylakoid membranes on the other hand contain F-type ATPase only. Our results emphasize the importance of plasma membrane energization even in obligately phototrophic cyanobacteria.