Dr. Linda Hufnagel
Cell and Molecular Biology
Tetrahymena, opsins, rhodopsins, bacteriorhodopsins, G-coupled protein receptors
Tetrahymena is a genus of ciliated protozoans, a diversified lineage of unicellular eukaryotes. They are freshwater organisms, and generally inhabit streams, lakes, and ponds. Tetrahymena thermophila is commonly studied as a model cell because of its unique variety of complex and specialized cell structures and processes, which are similar to those of higher animals.
G-protein-coupled receptors (GPCRs) are transmembrane proteins that transduce stimuli from outside of the cell into intracellular signals, through the interaction of their intracellular domains with heterotrimeric G proteins. GPCRs make up a vast protein family that includes a variety of subfamilies with distinct functions. They are usually described as a “superfamily” because they are made up of a group of families that have indications of evolutionary relationship but no significant similarities in sequences. Opsins are a subfamily of GPCRs; opsins include some that are found in the retina and are light-sensitive. Such “visual opsins” are involved in the transmission of signals from light to create visual images. Different types of photoreceptor cells contain different types of opsins. Rhodopsins are opsin family chemoproteins (proteins linked to a light-absorbing pigment such as retinal) that are found in membranous disks in the outer segment of rod cells and cone cells. Within the visual and non-visual opin family, there are many subdivisions that may be further subdivided into cone opsins and rhodopsins. These each have distinct molecular properties that are derived from the difference in the residues at position 122 and 189 of their amino acid sequences.
This project began with the hypothesis, that T. thermophila might contain rhodopsin-like proteins that absorb light and are involved in the light response. This hypothesis was based on two lines of evidence: first, experimental evidence that Tetrahymena can respond to light (Hufnagel and Kass-Simon, unpublished); and second, a prior genomic study that had accidently uncovered some preliminary evidence for rhodopsin-like proteins in T. thermophila (Babcock and Hufnagel, unpublished). With a more focused genomic search to identify rhodopsin- and opsin-like gene families in Tetrahymena, it was expected that opsin-like orthologues would be encountered. These then would be examined for evidence for a role in the light response. The focus of the project has been on the use of BLAST analysis of Tetrahymena genomic data to identify possible rhodopsin or opsin orthologues in Tetrahymena, and using expression libraries, BLAST analysis, motif search programs, and alignment methods to obtain clues about whether or not such opsin orthologues play a role in light reception. A diversified group of opsins were used to conduct gene blasting experiments to understand which visual opsins Tetrahymena is most closely related to. Further examination must be conducted in order to determine if Tetrahymenas have regions that can bind retinal-like molecules. So far, experimentation has only provided evidence for weakly orthologous proteins in Tetrahymena.
The methods used in the study will be described and the results obtained will be presented and their implications discussed.