Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii

The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.