Site-directed mutagenesis of the photosystem I reaction center in chloroplasts. The proline-cysteine motif.
Site-directed mutagenesis has been used to introduce specific amino acid changes into the photosystem I reaction center in the green alga Chlamydomonas reinhardtii. Plasmids containing mutated copies of the chloroplast psaB gene, encoding a polypeptide of the photosystem I reaction center heterodimer, were introduced into the chloroplast genome by particle bombardment. Successful transformants were selected by two procedures. The first involved complementation of a nonphotosynthetic mutant of Chlamydomonas, CC-2341 (ac-u-g-2.3), which has a frameshift mutation in the psaB gene, and selection of photosynthetic transformants on minimal medium. The second procedure utilized a co-transformation procedure with a plasmid containing a rRNA gene that confers spectinomycin resistance. Homologous replacement of the psaB gene was confirmed by screening for a unique restriction enzyme site within the transforming psaB sequences. These procedures have been used to specifically mutate a highly conserved proline-cysteine motif suggested to be important in coordinating the [4Fe-4S] iron-sulfur center Fx. Our results show that the cysteine is essential for assembly of the photosystem I reaction center although the adjacent proline fulfills no identifiable function. The approach described in this paper will be of value to future studies of the structure, function, and assembly of photosystem I.
Department of Botany, Arizona State University, Tempe 85287-1601.
A N
AN
Webber
P B
PB
Gibbs
J B
JB
Ward
S E
SE
Bingham
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D003545:Cysteine; D004247:DNA; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011392:Proline; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D004247:DNA; D011392:Proline; D003545:Cysteine