We have used a cross-linking approach to study the interaction of ferredoxin (Fd) with photosystem I (PSI). The cross-linking reagent 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.Chloroplast thylakoid membranes contain three integral membrane protein complexes which cooperate in the transfer of electrons from water to NADP. These are PSI, PSII, and the Cyt b6-f complex. In this noncyclic electron transport chain, PSI catalyzes light-induced electron transport from reduced plastocyanin to Fd (10).The PSI complex contains P700 as its reaction center Chl and a series of electron acceptors, Ao, A,, Fe-Sx, Fe-SA, and Fe-SB, which accept the electron lost from the reaction center Chl upon photooxidation (10,16 plexes. This cross-linker has previously been used to cross-link Fd to Fd:NADP+ reductase (20) and to cross-link plastocyanin to Cyt f (3). In this study, cross-linked products have been identified by immunoblotting with specific antibodies. Using this approach, a 22 kD subunit ofPSI has been identified as a possible Fd-binding protein in the PSI complex. MATERIALS AND METHODSThylakoid and PSI Preparation. Spinach (Spinacia oleracea) was grown hydroponically in a greenhouse. Washed, deribbed leaves were homogenized in blending buffer (50 mM Tris-HCl [pH 7.8], 0.3 M sucrose, 10 mM NaCl, 5 mM MgCl2). The homogenate was passed through filtering silk and centrifuged for 3 min at 3000 g. The pellets were resuspended in 10 mM MOPS buffer (pH 6.5) for use in cross-linking, or in blending buffer for activity assays. PSI-200 was prepared by the method of Mullet et aL (12). PSI-100 was prepared as described by Ortiz et al. (14). The preparations used in this work contained 185 Chl/P700 and 120 Chl/P700, respectively.Cross-Linking. Ferredoxin was cross-linked to thylakoids as described by Merati and Zanetti (I 1) with the following changes. The cross-linking reaction was carried out in 10 mM MOPS buffer (pH 6.5), 2 mM MgCl2 with 2.5 mm EDC. After quenching the reaction, blending buffer was used to dilute the membranes, which were then pelleted and resuspended in blending buffer.PSI-200 (0.5 mg Chl/mL) was incubated in the presence or absence of 30 jM Fd and 1 mm EDC in the MOPS-MgCl2 buffer f...

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.

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