Visualizing the mobility and distribution of chlorophyll proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation

The diffusion of proteins in chloroplast thylakoid membranes is believed to be important for processes including the photosystem-II repair cycle and the regulation of light harvesting. However, to date there is very little direct information on the mobility of thylakoid proteins. We have used fluorescence recovery after photobleaching in a laser-scanning confocal microscope to visualize in real time the exchange of chlorophyll proteins between grana in intact spinach (Spinacia oleracea L.) and Arabidopsis chloroplasts. Most chlorophyll proteins in the grana appear immobile on the 10-min timescale of our measurements. However, a limited population of chlorophyll proteins (accounting for around 15% of chlorophyll fluorescence) can exchange between grana on this timescale. In intact, wild-type chloroplasts this mobile population increases significantly after photoinhibition, consistent with a role for protein diffusion in the photosystem-II repair cycle. No such increase in mobility is seen in isolated grana membranes, or in the Arabidopsis stn8 and stn7 stn8 mutants, which lack the protein kinases required for phosphorylation of photosystem II core proteins and light-harvesting complexes. Furthermore, mobility under low-light conditions is significantly lower in stn8 and stn7 stn8 plants than in wild-type Arabidopsis. The changes in protein mobility correlate with changes in the packing density and size of thylakoid protein complexes, as observed by freeze-fracture electron microscopy. We conclude that protein phosphorylation switches the membrane system to a more fluid state, thus facilitating the photosystem-II repair cycle.