Mapping the lateral distribution of photosystem II and the cytochrome b6/f complex by direct immune labeling of the thylakoid membrane.

By direct immunolabeling we have mapped the distribution of photosystem II (PS II) and cytochrome b6/f on the surfaces of photosynthetic membranes isolated from spinach. Photosynthetic membranes were attached to a support and gently disrupted to expose the occluded outer stacked surface, prior to labeling. Polyclonal antibodies against PS II intensely labeled the outer stacked surfaces while the outer nonstacked surface had minimal labeling. This confirms previous fractionation and immunolocalization studies which demonstrated that PS II is largely restricted to the stacked regions of the membrane. Inside-out membranes were also heavily labeled with PS II antibodies. Antibodies against cytochrome f were evenly distributed between the stacked and nonstacked outer surfaces and were found clumped together on the membrane outer surface. Previous fractionation and immunolocalization studies have indicated that cytochrome b6/f is located in both the stacked and nonstacked regions, but this is the first report to provide direct evidence that the complex may be clustered in the membrane. The clustering of antibodies to cytochrome b6/f supports the idea that this electron transport component exists as a multimeric complex within the membranes and that such complexes are found in both stacked and nonstacked regions of the photosynthetic membrane. No evidence was seen of any special differentiation of the marginal regions of the membrane, which link stacked and nonstacked regions.