Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane

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Abstract
The transverse heterogeneity of the polypeptides associated with the photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation was studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS), and the proteolytic enzyme, Pronase E. The PSI reaction center polypeptide of .apprx. 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [14C]TNBS in unfractionated thylakoids. The 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [14C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these 2 subcomplexes from pronase-treated membranes. The 62, 19 and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at .apprx. 48 kilodaltons, and further proteolytic digestion generated 2 other fragments at 30 and 17 kilodaltons, respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI was developed.