Mass spectrometric identification of a PSI-LHCI-cytochrome b6f complex in both Arabidopsis stn7 and pph1

Abstract

Native PAGE separation of digitonin-solubilized thylakoid stroma lamellae resolves PSI and cytochrome b6f into separate bands. Mass spectrometric analysis of the 1D native gel bands showed that the bands are baskets of co-migrating complexes along the molecular size axis. Hierarchical clustering confirmed the separation of dimeric cytochrome b6f from PSI in 1D LN-PAGE. Interval-zone free-flow electrophoresis separated the same extracts by charge in a flowing buffer film, and the continuous fractionation indicated co-migration of complexes without the in-gel structural hindrances. A second-dimension LN-PAGE separated the complexes co-migrating in iZE into complexes with defined molecular size, allowing in-gel mass spectrometry identification in the gel bands. Both genotypes showed a 720 kDa fluorescent band in which cytochrome f (PetA) and cytochrome b6 (PetB) join the PSI-LHCI node (stn7, d = 0.50; pph1, d = 0.34). Lhcb1 and Lhcb2 are absent from the 720 kDa node. The 753 kDa band is specific to pph1 (d = 0.38). Phosphorylated LHCII1,2 joins PSI-LHCI there, and PetB is detected in every slice of that band without becoming a leaf of the tree.