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BIOENERGETICS AND PHOTOSYNTHESIS

Protein Diffusion and Macromolecular Crowding in Thylakoid Membranes^1,[W]

Institute of Botany, 48149 Munster, Germany (H.K., S.H.); School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom (J.F.A., C.W.M.); and Mathematics Institute, University of Warwick, Coventry CV4 7AL, United Kingdom (D.B.A.E.)

The photosynthetic light reactions of green plants are mediated by chlorophyll-binding protein complexes located in the thylakoid membranes within the chloroplasts. Thylakoid membranes have a complex structure, with lateral segregation of protein complexes into distinct membrane regions known as the grana and the stroma lamellae. It has long been clear that some protein complexes can diffuse between the grana and the stroma lamellae, and that this movement is important for processes including membrane biogenesis, regulation of light harvesting, and turnover and repair of the photosynthetic complexes. In the grana membranes, diffusion may be problematic because the protein complexes are very densely packed (approximately 75% area occupation) and semicrystalline protein arrays are often observed. To date, direct measurements of protein diffusion in green plant thylakoids have been lacking. We have developed a form of fluorescence recovery after photobleaching that allows direct measurement of the diffusion of chlorophyll-protein complexes in isolated grana membranes from Spinacia oleracea. We show that about 75% of fluorophores are immobile within our measuring period of a few minutes. We suggest that this immobility is due to a protein network covering a whole grana disc. However, the remaining fraction is surprisingly mobile (diffusion coefficient 4.6 ± 0.4 x 10^–11 cm² s^–1), which suggests that it is associated with mobile proteins that exchange between the grana and stroma lamellae within a few seconds. Manipulation of the protein-lipid ratio and the ionic strength of the buffer reveals the roles of macromolecular crowding and protein-protein interactions in restricting the mobility of grana proteins.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Helmut Kirchhoff (kirchhh{at}uni-muenster.de).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.115170

^* Corresponding author; e-mail kirchhh{at}uni-muenster.de.

Received December 18, 2007; accepted February 12, 2008; published February 20, 2008.

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