Mobility of chlorophyll-protein complexes in thylakoid membranes from plants

Conrad Mullineaux
Professor of Microbiology
School of Biological and Chemical Sciences
Queen Mary, University of London
Mile End Road,
London E1 4NS
UK

The aim is to study the mobility of protein complexes (including Photosystem II and LHCII) in plant thylakoid membranes. Lateral diffusion of these complexes must be important for regulation, biogenesis and repair of the membrane. However, it is problematic because the membrane is extremely crowded with proteins. How much does macromolecular crowding impede the mobility of thylakoid proteins?

The project was initiated in collaboration with Helmut Kirchhoff (then at Institut für Botanik, Münster, Germany, now at Washington State University). Our collaboration was funded by a Royal Society International Joint Project Grant. Further development of the work involved Tomasz Goral, a BBSRC research student jointly supervised by Alexander Ruban, and Prof David Epstein (Mathematics Institute, University of Warwick). Prof Epstein worked on the complex image analysis problems associated with this work. The aim is to extend the Fluorescence Recovery after Photobleaching (FRAP) techniques initially applied to the thylakoid membranes of cyanobacteria to thylakoid membranes from the chloroplasts of green plants. This is challenging because green plant thylakoids have a complicated structure and lateral heterogeneity.

Helmut Kirchhoff and Silvia Haferkamp devised a preparation of grana membranes from spinach which provides a simplified model system for studying protein mobility. We used FRAP to show that most of the chlorophyll-proteins in these grana membranes are immobile. However, a small proportion of the protein can diffuse surprisingly fast, suggesting that the complexes are organised so as to allow the rapid diffusion of "free" protein. This may be crucial for the rapid repair of damaged Photosystem II complexes in vivo. "Dilution" of the grana membranes with extra lipid allows direct assessment of the effects of macromolecular crowding on protein mobility.


FRAP image sequence showing mobility of chlorophyll-proteins in a patch of grana membrane (in this case "diluted" by fusion with liposomes). Chlorophyll fluorescence is imaged in a laser-scanning confocal microscope. A line is bleached across the membrane patch (position indicated by the arrow) and subsquent spread and recovery of the bleached line indicates diffusion of chlorophyll-protein complexes. See Kirchhoff et al (2008).

Tomasz Goral is involved in developing approaches for looking at protein mobility in intact thylakoid membranes from spinach and Arabidopsis. Use of Arabidopsis mutants will allow us to determine factors that influence protein mobility in intact thylakoid membranes. see Goral et al., 2010.


Schematic diagram illustrating one approach to measuring protein exchange between grana in intact thylakoid membranes. The confocal laser spot (shown in blue) is used to bleach chlorophyll fluorescence in a single granum. If chlorophyll-proteins can exchange between grana, this will be visualised as a recovery in fluorescence of the bleached granum, accompanied by a decrease in fluorescence of neighbouring grana.

Key publications on this topic:

Goral, T.K., Johnson, M.P., Brain, A.P.R., Kirchhoff, H., Ruban, A.V. and Mullineaux, C.W. (2010) Visualizing the mobility and distribution of chlorophyll-proteins in higher plant thylakoid membranes: effects of photoinhibition and protein phosphorylation Plant Journal 62, 948-959.

Kirchhoff, H., Haferkamp, S., Allen, J.F., Epstein, D.B.A. and Mullineaux, C.W. (2008) Protein diffusion and macromolecular crowding in thylakoid membranes. Plant Physiology 146, 1571-1578

Mullineaux, C.W. (2008) Factors controlling the mobility of photosynthetic proteins. Photochemistry and Photobiology 84, 1310-1316

Kana, R., Prášil, O.and Mullineaux, C.W. (2009) Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffusion in the lumen is very restricted. FEBS Letters 583, 670-674.