Spatial organization of the cytochrome b6- f complex within chloroplast thylakoid membranes

Abstract

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b 6- f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3′,5,5′-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b 6- f antibodies; (4) agglutination of membrane fragments with anti-Cyt b 6- f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b 6- f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b 6- f throughout all thylakoid membranes.