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

Chlamydomonas reinhardtii is a unicellular biflagellate green alga which has the ability to perceive external light and alter its swimming behavior in response. A specialized light sensing structure, the eyespot, is responsible for this sensory ability. The eyespot is composed of photoreceptors and signal transduction components thought to be localized to the plasma membrane of the cell, and an organized arrangement of carotenoid-filled lipid granules in the chloroplast underlying the plasma membrane. To identify the components involved in eyespot assembly, a screen for eyespot assembly mutants (Lamb, et al., 1999) identified four new loci deficient in eyespot assembly. Herein, we demonstrate the eyespot assembly mutants are generally capable photobehaviors, though the phenotype of the mutant strain affects the nature and manner in which the photobehaviors are affected. Additionally, we report on the isolation of a tagged allele of EYE2. Using DNA adjacent to the tag site, the EYE2 gene was isolated. Attempts to localize the protein in vivo were unsuccessful. The EYE2 gene encodes a protein which contains a putative thioredoxin active site motif. Transformants harboring a mutant EYE2 gene with the more C-terminal cysteine changed to serine possess eyespots and are phototactic. This substitution has only been observed to be tolerated in protein disulfide isomerases; we hypothesize that the function of EYE2 in eyespot assembly may be through the rearrangement of disulfide bonds in substrate proteins that function more directly in eyespot assembly