Part 3 Materials and Methods

Isolation of PSI Complexes from C. reinhardtii - The C. reinhardtii mutant psbD-His containing C-terminal fusion of a His6 tag with the D2 protein (14) was kindly donated by J. Minagawa (Hokkaido University, Sapporo, Japan). Cells were grown in Tris acetate-phosphate medium (15) to exponential growth phase. Thylakoid membranes were isolated from 20 liters of cultures by a procedure of Diner and Wollman (16) and resuspended in 400 mM sorbitol/5 mM Hepes-OH, pH 7.5/15 mM NaCl/10 mM MgCl2. LHCI-PSI supercomplexes were purified from the thylakoids by a modified method of Fischer et al. (17). Thylakoid membranes were harvested at 149,000 . g for 30 min, washed with 5 mM Tris-HCl, pH 7.5/15 mM NaCl/10 mM MgCl2, centrifuged as above, and resuspended in 5 mM Tris-HCl, pH 7.5 at a chlorophyll concentration of 0.8 mg/ml. The membranes were solubilized with 0.9% (w/v) .-D-dodecylmaltoside (DDM) on ice in the dark for 20 min and then centrifuged at 48,400 . g for 25 min to remove insoluble material. To remove Histagged PSII complexes, solubilized membranes were incubated with equilibrated nickel-nitrilotriacetic acid-agarose (Qiagen) at a 4:1 (v/v) ratio for 1 h in the dark. Partially PSII-depleted samples (unbound fraction) were subjected to fractionation on continuous sucrose density gradients prepared by the modified "freeze-thaw" method (18) with the interim buffer composed of 0.5 M sucrose/5 mM Tricine-NaOH, pH 8/0.05% DDM/0.5 M betaine. The LHCI-PSI supercomplexes were observed in the lowest, most dense green fraction of the gradient. LHCI complexes were purified as described in Croce et al. (19).

Characterization of Purified PSI Complexes - SDS-PAGE was carried out using the Tris-Tricine system described by Scha¨gger and von Jagow (20). Protein bands were resolved on 10% polyacrylamide gels in the presence of 6 M urea and visualized with Coomassie Brilliant Blue R-250 using standard procedures. For Western blotting, proteins separated on SDS-PAGE were transferred onto polyvinylidene difluoride membranes (BioRad) in 10 mM CAPS, pH 11/10% methanol using Mini-Trans-Blot apparatus (BioRad), according to the manufacturer's instructions. Blots were probed with polyclonal antibodies raised against maize LHCI complex and Chlamydomonas psa gene products, kindly donated by R. Bassi (University of Verona, Verona, Italy) and J.-D. Rochaix (University of Geneva, Geneva, Switzerland), respectively. C. reinhardtii Lhca3- and Lhcb1-specific polyclonal antisera were kindly provided by M. Hippler (University of Jena, Jena, Germany). Anti-Lhcb2 and anti-phosphothreonine polyclonal antisera were purchased from AgriSera and Zymed Laboratories Inc., respectively. Blots were developed with anti-rabbit IgG-horseradish peroxidase conjugate