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Molecular Plant Advance Access originally published online on November 28, 2008
Molecular Plant 2009 2(1):32-42; doi:10.1093/mp/ssn075
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© The Author 2008. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

Immunoelectron Microscopy for Locating Calvin Cycle Enzymes in the Thylakoids of Synechocystis 6803

Rachna Agarwala, Stefan Ortlebb, Jayashree Krishna Sainisa and Michael Melzerb,1

a Molecular Biology Division, Bhabha Atomic Research Center, Mumbai 400085, India
b Structural Cell Biology Group, Molecular Cell Biology Department, Leibniz Institute of Plant Genetics and Crop Plant Research, Correnstr. 3, Gatersleben 06466, Germany

1 To whom correspondence should be addressed. E-mail melzer{at}ipk-gatersleben.de, fax 0049-39482/5500.

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/oxygenase (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) +ADP dependent CO2 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.

Key Words: High-pressure freezing • immunogold labeling • membrane-isolation • Synechocystis • thylakoids


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