Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane 1 STUDIES OF PHOTOSYSTEM I USING A CHEMICAL PROBE AND PROTEOLYTIC

The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of -62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by I'4CFTNBS in unfractionated thylakoids. On the other hand, 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 I"4CITNBS 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 two subcomplexes from pronasetreated 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 about 48 kilodaltons, and further proteolytic digestion generated two 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 has been developed. complexes to the overall structure of the chloroplast membrane is less well understood. It has recently been possible to isolate all the chloroplast membrane electron transfer complexes in highly resolved functional form (7, 16, 25), and subsequently it has been demonstrated that these complexes are reconstitutively active in catalyzing noncyclic electron transport from water to NADP when supplemented with soluble protein cofactors (19). The availability of these resolved complexes allows for detailed examination of structure-function relationships in the individual complexes and examination of the nature of the interactions between complexes. In the present study, we have considered the topographical orientation of the chloroplast PSI complex. The impermeant chemical probe, TNBS,3 labeled with "1C, has been used to modify stroma-exposed amino acid side chains on proteins in unfractionated thylakoid membranes and reactive groups in the resolved PSI complex of Mullet et al. (25). The labeling pattern of PSI polypeptides in the membranes has been compared with that of the isolated PSI complex and subfractions isolated from this complex. Since any one specific technique used in the study of membrane-protein topology has limitations, different techniques should be applied before drawing conclusions on membrane organization. To that effect, we have used proteolytic digestion with pronase E to probe the PSI complex for components accessible to the stroma phase. This approach has been previously employed in studies of the organization of mitochondrial (5) and chromatophore (29) membrane proteins as well as in the characterization of thylakoid pigment-protein complexes (3, 10). Based on these results, a model for the PSI polypeptides that shows a transverse heterogeneity with respect to the membrane is presented and the organization of the thylakoid PSI complex is considered. Thylakoid membranes in higher plants contain four integral protein complexes: PSI, PSII, the Cyt b6-fcomplex, and the ATPsynthetase (CFo-CF,) (17). These complexes function in a series of reactions that involve the absorption of photons and the conversion of this light energy into the stable chemical products, ATP and NADPH, that are ultimately used for the fixation of CO2 via the reductive pentose phosphate cycle. Although the function of these individual complexes has been studied in great detail, the nature of the organization of the individual components in the respective complexes and the relationship of these 'Supported in part by a grant from the National Science Foundation (PCM-84