Opsins are seven-transmembrane helix proteins that bind all-trans or 11-cis isomers of retinal to form light-absorbing pigments known as rhodopsins. Previously, opsin-encoding genes had only been cloned from animals and the archaea. The principal investigator recently identified the first opsin gene from eukaryotic microbes, Neurospora crassa nop-1. The NOP-1 protein sequence is most similar to that of archaeal opsins, with conservation of all 22 retinal binding pocket residues, including the lysine residue that forms a Schiff base linkage with retinal. NOP-1 is also similar to several predicted proteins from various fungal species, including Saccharomyces cerevisiae Hsp30p. With the exception of two predicted proteins from filamentous fungi, all other related fungal proteins lack the Schiff base lysine residue and we have referred to them as Opsin-Related Proteins (ORPs). NOP-1 has been overexpressed in Pichia pastoris and its spectral properties determined. Similar to archaeal opsins, NOP-1 binds all-trans retinal with a Schiff base linkage. The resultant pigment has an absorption maximum at 534 nm (green). The relatively long photocycle of the NOP-1 pigment is similar to that of archaeal sensory rhodopsins, suggesting NOP-1 functions as a sensory receptor in N. crassa. The nop-1 message is most abundant under conditions that favor asexual sporulation (conidiation) in N. crassa. Although ?nop-1 mutants do not have visible phenotypes at 30oC, these strains exhibit green light-dependent defects in asexual spore-forming structures and cell viability at elevated growth temperatures (37-42oC). Also, the principal investigator identified an EST encoding a N. crassa ORP (orp-1). The predicted ORP-1 protein sequence is most similar to HSP30 from Coriolus versicolor. In contrast to NOP-1, ORP-1 only shows conservation of 50% of the 22 retinal binding pocket residues with archaeal rhodopsins, including substitution of isoleucine for the Schiff base lysine residue. The sequence similarity between NOP-1 and archaeal rhodopsins; the evolutionary relationship between NOP-1 and S. cerevisiae Hsp30p; the light-dependent expression of nop-1; the sensory rhodopsin-like photocycle of NOP-1; the light-dependent conidiation defect of ?nop-1 mutants in the presence of oligomycin, and the light-dependent defects in cell growth at elevated temperatures lead to the following hypothesis: Opsins and ORPs regulate cell growth, viability and development during cellular stress responses in N. crassa, potentially via light-dependent and independent pathways, respectively. The Research Objectives of this project are 1) To determine the localization and native chromophore of NOP-1 in N. crassa and to further characterize the involvement of nop-1 in stress and developmental regulation; 2) to identify genes that regulate nop-1 gene expression and genes whose expression is regulated by nop-1; 3) to create and analyze a ?orp-1 mutant and to over-express and characterize the encoded ORP-1 protein, and 4) to identify other genes encoding opsins and ORPs, and other nop-1 pathway components. During this collaborative research project, one laboratory will focus on Objectives 1 and 3, while the other will focus on Objectives 2 and 4. This collaborative research project will strengthen and extend the existing collaboration, and exploit the complementary skills and resources available to the two principal investigators. One principal investigator has extensive expertise in genome analysis, molecular evolution, the genetics and biochemistry of stress responses and the genetics of filamentous fungi in general. The other has significant experience in protein biochemistry, mutational analysis of fungal genes, stress responses and signal transduction via both G proteins and histidine kinases. The former has access to the resources of the Neurospora Genome Project, including automated sequencing and microarray facilities. The latter is in close proximity to a leader in biophysical analysis of archaeal rhodopsins and who has been a past and is a current collaborator. The existence of seven-helix opsin receptors has been postulated in eukaryotic microbes for decades. Many of these organisms utilize light signals to regulate cell growth, reproduction, phototaxis or the circadian clock. Therefore, the identification of an opsin in N. crassa has enormous repercussions for filamentous fungi and other eukaryotes. Furthermore, the lack of an opsin in the yeast Saccharomyces cerevisiae points to the importance of studying this class of proteins in the model filamentous fungus N. crassa. In addition, very little is known about ORPs in fungi: for example, whether they can bind a chromophore, absorb light, etc. Thus, the principal investigators are in the position of making pivotal discoveries in this field using N. crassa as an experimental organism.
