Molecular crowding and order in photosynthetic membranes Helmut Kirchhoff Institute of Botany, Schlossgarten 3, D-48149 Mu ¨ nster, Germany The integrity and maintenance of the photosynthetic apparatus in thylakoid membranes of higher plants requires lateral mobility of their components between stacked grana thylakoids and unstacked stroma lamel- lae. Computer simulations based on realistic protein densities suggest serious problems for lateral protein and plastoquinone diffusion especially in grana mem- branes, owing to strong retardation by protein com- plexes. It has been suggested that three structural features of grana thylakoids ensure efficient lateral transport: the organization of protein complexes into supercomplexes; the arrangement of supercomplexes into structured assemblies, which facilitates diffusion process in crowded membranes; the limitation of the diameter of grana discs to less than 500 nm, which keeps diffusion times short enough to support regula- tion of light harvesting and repair of photodamaged photosystem II. Introduction: lateral diffusion in thylakoid membranes In higher plants, photosynthetic energy conversion is associated with a structurally complex biomembrane that has fascinated researchers for many decades. Details of the architecture of the thylakoid membranes inside chloro- plasts are now available not only at the level of single protein complexes [1] but also at the level of the large-scale organization of the membrane (i.e. the complex folding of the membrane, which generates tightly appressed grana stacks) [2]. The structural consequence of grana formation is a subdivision of the membrane into stacked regions, comprising 80% of the membrane [3], and unstacked regions (known as stroma lamellae). This results in a non-uniform distribution of photosynthetic protein com- plexes [3]. Photosystem II (PSII) and light-harvesting complex II (LHCII) are concentrated in grana stacks, whereas most of the PSI, LHCI and the ATPase complexes are localized in unstacked membrane regions. This asym- metric distribution of proteins implies that protein com- plexes from stacked grana regions have to diffuse up to several hundred nm to reach unstacked stroma lamellae. Protein traffic between thylakoid subcompartments is a prerequisite for adaptation to environmental challenges as well as for repair and biogenesis of the photosynthetic apparatus. Rates of protein turnover in thylakoid mem- branes are particularly high. For example PSII in grana thylakoids is frequently damaged by light and regenerated by a sophisticated multi-step repair cycle [4]. In the course of this cycle – its half-time is shorter than one hour – photodamaged PSII complexes move out of the grana to the stroma lamellae, where the repair machinery is loca- lized. The regenerated complex is then transferred back to stacked grana regions. Furthermore, it has long been known that the distribution of light absorption between the two photosystems (i.e. PSI and PSII) is highly regulated [5]. In the course of this process (known as state transition), a fraction of LHCII can be redistributed be- tween PSII in the grana and PSI in unstacked membrane regions. This is triggered by reversible protein phosphoryl- ation through redox-controlled kinases. Thus, the function- ality and structural integrity of the photosynthetic machinery in higher plant thylakoid membranes requires efficient lateral protein diffusion. Moreover, since the ‘Z scheme’ of photosynthetic elec- tron flow was introduced [6], it has become clear that, in addition to intramolecular electron transfer, intermolecu- lar reactions among the large building blocks of PSII, the cytochrome b 6 f (cyt b 6 f ) complex and PSI are required for energy conversion. These diffusion-dependent electron transfer reactions are managed by small electron carriers; plastoquinone moves in the hydrophobic lipid phase of the membrane bilayer (between PSII and cyt b 6 f complex), and plastocyanin moves in the aqueous thylakoid lumen (be- tween cyt b 6 f complex and PSI). Remarkably, out of 20 photosynthetic electron transfer reactions, only the two mentioned above are diffusion-dependent. A similar ratio between intramolecular and intermolecular electron trans- fer reactions exists for the respiratory electron transfer chain in mitochondria. This might indicate an evolutionary pressure to reduce to a minimum the diffusion-dependent redox reactions in bioenergetic membranes. Today, 50 years since the Z scheme was postulated, it is still not clear how lateral diffusion works in thylakoid membranes. There might be two main reasons for this lack of under- standing. On the one hand, we don’t have a precise mol- ecular picture of the organization and dynamics of protein complexes in thylakoids. On the other hand, there is a complex dependence of lateral diffusion processes on the membrane architecture. It is known that the arrangement, size and packing of protein complexes are crucial parameters for lateral membrane transport [7]. The situ- ation in thylakoid membranes is particularly complex, because the protein organization is not static but highly dynamic, depending on environmental factors. Estimates of the protein-packing density show that grana thylakoids are among the most crowded biomembranes in nature. This has strong implications for lateral diffusion processes Opinion Corresponding author: Kirchhoff, H. (kirchhh@uni-muenster.de). 1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.03.001 Available online 11 April 2008 201