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Intact spinach (Spinacia oleracea) thylakoid membranes were treated with various proteases and photosystem I (PSI) complexes were isolated from these membranes to define the membrane topology of specific PSI subunits. Trypsin treatment caused cleavage of the PSI-D and E subunits. Thermolysin treatment cleaved the PSI-D, E, H, and K subunits, and also caused limited degradation of the reaction center core PSI-A and B subunits. Pronase treatment produced the most dramatic results as the PSI-A and B subunits were cleaved to 47-, 45-, 26-, and 24-kilodalton products. In addition, pronase degraded the PSI-D, E, H, K, and L subunits. Proteolytic cleavage sites for several of the products were identified by amino acid sequencing. The results indicate that PSI-A, B, D, E, H, K, and L subunits all have stroma-exposed regions, and these findings are summarized in a model describing the subunit organization of PSI.

We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent N-ethyl-3-(3-dimethylaminopropyl) carbodiimide was found to cross-link spinach Fd to a 22 kilodalton subunit of PSI in both isolated spinach (Spinacia oleracea) PSI complexes and spinach thylakoid membranes. The product had an apparent molecular weight of 38 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified as a cross-linked product using specific antibodies to Fd and the 22 kilodalton subunit. In both a native PSI complex (200 Chl/P700) and a PSI core complex (100 Chl/P700), a second cross-linked product at 36 kilodaltons was seen. The latter cross-reacted with an antibody to Fd but did not cross-react with antibodies directed against the 24.3, 22, 19, 17.3 or 8.5 kilodalton, or psaC subunits of PSI. Its composition remains to be determined. In thylakoids only the 38 kilodalton product was observed along with a cross-linked complex of Fd and Fd:NADP(+) reductase.