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The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays. Whereas EYE3, a serine/threonine kinase of the ABC1 family, localizes to pigment granules, EYE2 localization corresponds to an area of the chloroplast envelope in the eyespot. EYE2 is positioned along, and adjacent to, the D4 rootlet in the absence of pigment granules. The eyespot pigment granule array is required for maintenance of the elliptical shape of both the overlying EYE2 and channelrhodopsin-1 photoreceptor patches. We propose a model of eyespot assembly wherein rootlet and photoreceptor direct EYE2 to an area of the chloroplast envelope, where it acts to facilitate assembly of pigment granule arrays, and EYE3 plays a role in the biogenesis of the pigment granules.

EYE2 is a key protein in connecting the positioning information of the microtubule rootlet cytoskeleton and channelrhodopsin 1 (ChR1) photoreceptor to the formation and positioning of the eyespot pigment granules in the chloroplast of Chlamydomonas. EYE3, a ser/thr kinase of the ABC1 family, is found in pigment granules and is required for their biogenesis.

ABSTRACTAssembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtiirequires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1(mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in CHLAMYDOMONAS: In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.