Immunoelectron microscopy for locating Calvin cycle enzymes in the thylakoids of synechocystis 6803

Abstract

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing (HPF) and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle (phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxy-genase (RuBisCO), 3- phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase) and the catalytic portion of the chloroplast H+-ATP synthase (CF1) are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate (R-5-P) 1ADP dependent CO 2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.