Cloning and Sequencing of a Full-Length cDNA Clone Encoding the PSI-D Subunit of Photosystem I from Barley

PSI catalyzes the electron transport from reduced plastocyanin to oxidized Fd in higher plants and cyanobacteria. Thirteen polypeptides have been identified in the PSI complex from higher plants (Andersen and Scheller, 1993). Five of the polypeptides are chloroplast encoded (PSI-A, -B, -C, -I, -J) and eight are nuclear encoded (PSI-D, E , -F, -G, -H, -K, -L, -N). The psaA and psaB genes encode the two major polypeptides in the PSI complex that bind pigments, the reaction center P700 and the electron acceptors Ao, AI, and X. The PSI-C subunit binds the terminal electron acceptors A and B (Hdj et al., 1987). Cross-linking experiments have shown that the polypeptide PSI-D encoded by the PsaD gene interacts with Fd (Zanetti and Merati, 1987). A cDNA clone encoding PSI-D from spinach has previously been isolated (Lagoutte, 1988). The primary structure of the transit peptide and the hydrophilic character of PSI-D predict an extrinsic polypeptide located on the stromal side of the thylakoid membrane. Extraction of PSI-D from the PSI core with nbutanol (Oh-oka et al., 1988) supports this assignment. Reconstitution experiments with PSI-D and PSI-C overexpressed in Escherichia coli have shown that PSI-C binding to the PSI-A/PSI-B heterodimer in vitro requires the presence of PSI-D (Li et al., 1991). A cDNA library of poly(A)+ RNA from light-induced barley (Hordeum vulgare L.) seedlings was constructed in the h ZAP I1 vector (Stratagene, La Jolla, CA). The library was screened with a 5'-end-labeled oligonucleotide specifying the barley PSI-D (Okkels et al, 1988). A 637-bp long partial cDNA clone was identified. This partial clone was used as a probe in subsequent screens. The insert sizes of 30 positive clones were determined by Southern blotting. Inserts from five possible full-length clones were in vivo excised from the h phage using the helper phage M13K07 (Short et al., 1988). Sequencing showed that one clone of 835 bp was a fulllength cDNA clone (Fig. 1, Table I). The cDNA contains an open reading frame of 618 bp. The amino acid sequence deduced from the cDNA clone contains regions that match the partial amino acid sequences obtained (Scheller et al., 1988). The deduced amino acid sequence also shows high similarity to PSI-D from other plants and cyanobacteria. The