Two dimensional electrophoresis of thylakoid membrane proteins and its application to microsequencing

A procedure of two-dimensional gel electrophoresis adapted for application on membrane proteins from the thylakoids is described. It involves isoelectric focusing in the first dimension and size dependent electrophoresis in the second dimension. About 100 polypeptides are clearly separated with relatively little streaking. About 20 polypeptides are identified by immunoblotting or location in the gel. They are the polypeptides of the PS I core, the 64 kDa protein, the α and β subunits of CF1 ATPase, cytochrome f, Rieske iron-sulfur protein, the 23 kDa and 33 kDa polypeptides of the oxygen evolving complexes, CP29, CP24, CP27 and CP25 (last two proteins belong to LHCII). Some proteins give rise to two or more separate spots indicating a separation of different isoforms of these proteins. Among them, the LHCII polypeptides (27 kDa and 25 kDa) were each resolved into at least three spots in the pH range 4.75-5.90; the Rieske FeS protein, as published elsewhere (Yu et al. 1994), was separated into two forms having different isoelectric points (pI 5.1 and 5.4), each of them was also microsequenced; the 64 kDa protein claimed to be a LHCII-kinase was found to be multiple forms appearing in at least two isoforms with pI 6.2 (K1) and 6.0 (K2) respectively, furthermore, K1 can be resolved into two subpopulations.The lateral distribution of these proteins in the thylakoid membrane was determined by analysing the vesicles originating from different parts of the thylakoids. The data obtained from this analysis can be partially used as markers for different thylakoid domains.This procedure for sample solubilization and 2-D electrophoresis is useful for the analysis of the polypeptide composition of vesicles originating from the thylakoid membrane and for microsequences of individual polypeptides isolated from the 2-D gel.