Intragenic enhancers and suppressors of phytoene desaturase mutations in Chlamydomonas reinhardtii
Phoi T. Tran, Marina N. Sharifi, Subhajit Poddar, Marilyn C. Kobayashi, Rachel M. Dent, and Krishna K. Niyogi
Dept. of Plant and Microbial Biology, Univ. of CA, Berkeley, 111 Koshland Hall, Berkeley, CA 94720, USA
Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating point mutant, pds1-1, was isolated in Chlamydomonas reinhardtii, and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis - the conversion of phytoene to ζ-carotene. The pds1-1 mutant is leaky for PDS activity as it still accumulated downstream colored carotenoids. The pds1-1 mutant was mutagenized again to isolate enhancers that completely lacked PDS activity. A white double mutant (pds1-2), which is null for PDS activity, was isolated in this screen. A second null mutant was also identified from an insertional mutagenesis screen. Both null mutants only accumulated phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced fitness compared to wild-type cells and white psy mutants, which affect phytoene synthase. Insight into amino acid residues important for PDS activity was obtained through the characterization of 16 pds1-2 suppressor mutants. The pds1-2 suppressor mutants fell into three classes: four were revertants of the pds1-1 point mutation, seven had mutations that changed PDS amino acid residue Pro64 to Phe, and five had mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS, suggest that amino acids at positions 64 and 90 are in close contact in the active PDS protein and have important roles in its structural stability and activity.
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