Probing the dynamics of photosynthetic membranes with fluorescence recovery after photobleaching.

In the past few years, there has been remarkable progress in knowledge of the structures and organization of the protein complexes of photosynthetic membranes. However, static structures do not tell the whole story. Photosynthetic membranes, like other biological membranes, are dynamic systems. Recent technological advances are making it increasingly easy to probe the dynamics of photosynthetic membranes using fluorescence recovery after photobleaching. Here we explain the potential and the limitations of the technique.

Lipid diffusion in the thylakoid membranes of the cyanobacterium Synechococcus sp.: effect of fatty acid desaturation.

Thylakoid membranes are crucial to photosynthesis in cyanobacteria and plants. In cyanobacteria, genetic modification of membrane lipid composition strongly influences cold tolerance and susceptibility to photoinhibition. We have used fluorescence recovery after photobleaching to measure the diffusion of a lipid-soluble fluorescent marker in cells of the cyanobacterium Synechococcus sp. PCC 7942. We have compared the wild-type strain with a transformant with an increased level of fatty acid unsaturation. The transformant showed a six-fold increase in the diffusion coefficient for the fluorescent marker at growth temperature. This is the first direct measurement of lipid diffusion in a photosynthetic membrane.

Mobilization of photosystem II induced by intense red light in the Cyanobacterium Synechococcus sp PCC7942.

We use confocal fluorescence microscopy and fluorescence recovery after photobleaching to show that a specific light signal controls the diffusion of a protein complex in thylakoid membranes of the cyanobacterium Synechococcus sp PCC7942 in vivo. In low light, photosystem II appears completely immobile in the membrane. However, exposure to intense red light triggers rapid diffusion of up to approximately 50% of photosystem II reaction centers. Particularly intense or prolonged red light exposure also leads to the redistribution of photosystem II to specific zones within the thylakoid membranes. The mobilization does not result from photodamage but is triggered by a specific red light signal. We show that mobilization of photosystem II is required for the rapid initiation of recovery from photoinhibition. Thus, intense red light triggers a switch from a static to a dynamic configuration of thylakoid membrane protein complexes, and this facilitates the rapid turnover and repair of the complexes. The localized concentrations of photosystem II seen after red light treatment may correspond to specific zones where the repair cycle is active.

Mobility of the IsiA chlorophyll-binding protein in cyanobacterial thylakoid membranes.

We are using fluorescence recovery after photobleaching (FRAP) to probe the dynamics of thylakoid membranes in vivo in cells of the cyanobacterium Synechococcus sp. PCC7942. We have shown previously that the light-harvesting phycobilisomes diffuse quite rapidly on the thylakoid membrane surface. However, the photosystem II core complexes appear completely immobile. This raises the possibility that all of the membrane integral protein complexes in the thylakoid membrane are locked into a rather rigid array. Alternatively, it is possible that photosystem II is specifically anchored in the membrane, with other membrane proteins able to diffuse around it. We have now resolved this question by studying the diffusion of a second integral membrane protein, the IsiA chlorophyll-binding protein. IsiA is induced under iron starvation and some other stress conditions. In iron-stressed cyanobacterial cells, a high proportion of chlorophyll fluorescence comes from IsiA. This makes it straightforward to examine the diffusion of IsiA by FRAP. We find that the complex is mobile with a mean diffusion coefficient of approximately 3 x 10(-11) cm(2) s(-1). Thus it is clear that some thylakoid membrane proteins are mobile and that there must be a specific anchor that prevents photosystem II diffusion. We discuss the implications for the structure and function of the cyanobacterial thylakoid membrane.

Phycobilisome Mobility in the Cyanobacterium Synechococcus sp. PCC7942 is Influenced by the Trimerisation of Photosystem I.

We have constructed a mutant of the cyanobacterium Synechococcus sp. PCC7942 deficient in the Photosystem I subunit PsaL. As has been shown in other cyanobacteria, we find that Photosystem I is exclusively monomeric in the PsaL(-) mutant: no Photosystem I trimers can be isolated. The mutation does not significantly alter pigment composition, photosystem stoichiometry, or the steady-state light-harvesting properties of the cells. In agreement with a study in Synechococcus sp. PCC7002 [Schluchter et al. (1996) Photochem Photobiol 64: 53-66], we find that state transitions, a physiological adaptation of light-harvesting function, occur significantly faster in the PsaL(-) mutant than in the wild-type. To explore the reasons for this, we have used fluorescence recovery after photobleaching (FRAP) to measure the diffusion of phycobilisomes in vivo. We find that phycobilisomes diffuse, on average, nearly three times faster in the PsaL(-) mutant than in the wild-type. We discuss the implications for the mechanism of state transitions in cyanobacteria.

Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803.

The transport and sorting of extracytoplasmic proteins in cyanobacteria is made complex by the presence of a highly differentiated membrane system. Proteins destined for the periplasm and thylakoid lumen are initially transported by Sec- and Tat-type pathways but little is known of the mechanisms that ultimately direct them to the correct destinations. We have generated a Synechocystis PCC6803 transformant that expresses a fusion protein comprising the Tat-specific targeting signal of Escherichia coli TorA linked to green fluorescent protein (GFP). Immunoblotting indicates the presence of mature-size GFP but no precursor form, demonstrating that efficient translocation has taken place. Confocal microscopy and immunogold electron microscopy reveal GFP to be almost exclusively located in the periplasm, with almost no protein evident in the thylakoid network. These data point to the operation of highly effective sorting pathways for soluble proteins in this cyanobacterium. The observed sorting of the GFP suggests that either (a) the Tat apparatus is located only in the plasma membrane or (b) the TorA-GFP is targeted across either membrane but the GFP is subsequently directed to the periplasm, perhaps by a default sorting pathway to this compartment.