Isolation and characterization of cDNA clones encoding the 17.9 and 8.1 kDa subunits of Photosystem I from Chlamydomonas reinhardtii

Abstract

cDNA clones encoding two Photosystem I subunits of Chlamydomonas reinhardtii with apparent molecular masses of 18 and 11 kDa (thylakoid polypeptides 21 and 30; P21 and P30 respectively) were isolated using oligonucleotides, the sequences of which were deduced from the N-terminal amino acid sequences of the proteins. The cDNAs were sequenced and used to probe Southern and Northern blots. The Southern blot analysis indicates that both proteins are encoded by single-copy genes. The mRNA sizes of the two components are 1400 and 740 nucleotides, respectively. Comparison between the open reading frames of the cDNAs and the N-terminal amino acid sequences of the proteins indicates that the molecular masses of the mature proteins are 17.9 (P21) and 8.1 kDa (P30). Analysis of the deduced protein sequences predicts that both subunits are extrinsic membrane proteins with net positive charges. The amino acid sequences of the transit peptides suggest that P21 and P30 are routed towards the lumenal and stromal sides of the thylakoid membranes, respectively.

Insertion of the N-terminal Part of PsaF from Chlamydomonas reinhardtii into Photosystem I from Synechococcus elongatus Enables Efficient Binding of Algal Plastocyanin and Cytochrome c 6

“…1). A DNA construct, pCRSEF/2, was constructed in which the region of the psaF gene from S. elongatus encoding the mature PsaF subunit was substituted completely by the psaF region from C. reinhardtii (30). First, an EcoRV site was introduced at codons 24 and 25 of psaF (i.e.…”

“…These modifications were introduced by inverse PCR in the presence of oligonucleotides SEF1 (5Ј-TGCGATAT-CAGCGGAGGCTAGG-3Ј) and SEF2 (5Ј-TCTCTAGATTTGCTGTTT-GTTG-3Ј) to create the vector pSEF1. Second, a psaF cDNA clone from C. reinhardtii (30) was modified by PCR in the presence of oligonucleotides CRF1 (5Ј-CCGATATCGCGGGCCTGACC-3Ј) and CRF2 (5Ј-CAGCTAGCGGGGAGACACGG-3Ј) to create an EcoRV site at codons 63 and 64 (i.e. codons 1 and 2 of the mature PsaF) and an NheI site at the termination codon.…”

The N-terminal domain of PsaF: Precise recognition site for binding and fast electron transfer from cytochromec6and plastocyanin to photosystem I ofChlamydomonas reinhardtii

“…1), found to be involved in the crosslink of pc to PSI from spinach, is also conserved in the PsaF protein of C. reinhardtii (37). In particular, Lys12, Lys16, Lys19, or Lys23 within this ␣-helix motif of PsaF were suggested to interact with the conserved acidic patch 42-44 of pc (10).…”

Polypeptide composition of higher plant photosystem I complex

“…'Native' PSI complex which additionally retains LHCI can be isolated from green plants by a milder detergent treatment, while cyanobacterial PSI has no LHCI-like antenna. Until recently the subunit proteins found in these various types of PSI complexes from both green plants and cyanobacteria have been classified in three categories as follows: (i) two chlorophyll a-binding subunits of about 80 kDa, which carry P700, acceptors A0 and A1, and Fe-S center X, (ii) 5-6 core subunits ranging from 8 to 22 kDa which include Fe-S center A/B protein, plastocyanin-docking protein and ferredoxin-docking protein, (iii) 4-5 LHCI subunits of Abbreviations: CPI, chlorophyll-protein complex 1 of photosystem I reaction center; LHCI, light-harvesting chlorophyll complex associated with photosystem I; PSI, photosystem I; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis green plants ranging from 15 to 25 kDa which bind antenna chlorophyll a and b. Sequencing of these proteins and/or their corresponding genes have registered two genes, psaA and psaB, for the P700-carrying proteins [2,3], 6 genes, psaC to psaH, for the core subunits [4][5][6][7][8][9][10][11][12][13][14][15][16][17] and at least three types of cab genes for the LHCI subunits [18][19][20]. In green plants, psaA, psaB and psaC are encoded by chloroplast genome while the others are encoded by nuclear genome.…”