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Title: DIRECTED MUTAGENESIS OF CHLOROPLAST RUBISCO: ANALYSIS OF THE RUBSICO ACTIVASE-BINDING REGION

Author
item O'BRIEN, CAROLYN - BIOCHEMISTRY UNIV NEB
item PORTIS JR, ARCHIE
item SPREITZER, ROBERT - BIOCHEMISTRY UNIV NEB

Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only
Publication Acceptance Date: 7/1/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Rubisco requires a second chloroplast protein, Rubisco activase, to facilitate active-site carbamylation and maximal enzyme activity. Activase proteins from different species display specificity for particular Rubisco enzymes. It has recently been shown that a P89R substitution in the chloroplast-encoded large subunit of Chlamydomonas reinhardtii Rubisco changes the specificity of the interactions between Chlamydomonas rubisco and Rubisco activase from higher-plant species. Thus, an activase-binding region has been defined on the surface of the Rubisco large subunit. However, the P89R mutant strain has a wild-type (photosynthesis-competent) phenotype. In an attempt to disrupt the interaction between Rubisco and activase, we have used directed mutagenesis to create P89A and P89W substitutions. Surprisingly, the P89A substitution also produces a wild-type phenotype via chloroplast transformation, and the P89A mutant strain has a normal level of Rubisco protein. In contrast, photosynthesis-competent transformants cannot be recovered with the P889W substitution. Work is in progress to screen for photosynthesis-deficient, acetate-requiring transformants via a co-transformation method. If P89W blocks the interaction between Rubisco and activase, it may be possible to recover compensatory activase mutants by selecting for photosynthesis-competent revertants. Other large-subunit substitutions have been made in the activase-binding region (D86R and D98K), but they also fail to eliminate photosynthesis. Work is in progress to characterize the interactions between these mutant enzymes and Rubisco activase.