Site-directed mutagenesis of the photosystem I reaction center in chloroplasts: The proline-cysteine motif

Andrew Webber, Pamela B. Gibbs, Joni B. Ward, Scott E. Bingham

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)12990-12995
Number of pages6
JournalJournal of Biological Chemistry
Volume268
Issue number17
StatePublished - Jun 15 1993

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Photosystem I Protein Complex
Mutagenesis
Chloroplasts
Site-Directed Mutagenesis
Proline
Cysteine
Genes
Plasmids
Chloroplast Genes
Chloroplast Genome
Spectinomycin
Chlamydomonas
Chlamydomonas reinhardtii
Frameshift Mutation
Chlorophyta
Gene encoding
Algae
rRNA Genes
Sulfur
Screening

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of the photosystem I reaction center in chloroplasts : The proline-cysteine motif. / Webber, Andrew; Gibbs, Pamela B.; Ward, Joni B.; Bingham, Scott E.

In: Journal of Biological Chemistry, Vol. 268, No. 17, 15.06.1993, p. 12990-12995.

Research output: Contribution to journalArticle

Webber, Andrew ; Gibbs, Pamela B. ; Ward, Joni B. ; Bingham, Scott E. / Site-directed mutagenesis of the photosystem I reaction center in chloroplasts : The proline-cysteine motif. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 17. pp. 12990-12995.
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