Abstract
Abstract: :
Purpose: We previously identified an outer segment localization signal in the C-terminus of peripherin (ARVO 2001). We have now further characterized some of the functional properties of this peptide sequence. Methods: GFP fusion proteins containing various portions of the C-terminus of Xrds38 (the X. laevis homolog of peripherin) were expressed in rod photoreceptors of transgenic X. laevis under the control of the Xenopus opsin promoter. Frozen retinal sections were obtained from stage 48-60 eyes fixed in 4% paraformaldehyde. Localization of the fusion proteins was determined by confocal fluorescence microscopy. Electron microscopy was performed on plastic embedded retinas to analyze the localization and physiological effects of the fusion proteins. A COS cell expression system was used to probe for peripherin-peripherin interactions. Results: Our previous work demonstrated that the C-terminal 65 amino acids of peripherin were sufficient to direct a membrane bound GFP (XperCT282-347) exclusively to the rod outer segment (ROS). The ROS targeting signal was then further refined to the terminal 30 amino acids (XperCT317-347). Although both fusion proteins were targeted to the ROS, XperCT282-347 but not XperCT317-347 partially localized to incisures. A series of other fusion proteins were expressed which contained deletions extending towards either the distal C-terminus or the juxtamembrane region. Analysis of the localization of these fusion proteins allowed us to map the region responsible for incisure localization. Because peripherin is know to interact with itself to create higher order oligomers, we co-expressed GFP-bovine peripherin and GFP-bovine peripherin CT in COS cells. Immunoprecipitation of peripherin from cell lysates did not reveal any interaction between the C-terminal fusion protein and full-length peripherin. Immuno-electron microscopy showed that XperCT282-347 localization only partially overlapped with rhodopsin in the Golgi. Conclusion: The C-terminal of peripherin contains distinct ROS and incisure localization signals. Our results suggest that XperCT282-347 interacts with another disk rim/incisure protein (that is not full length peripherin) during biosynthesis/transport. EM immunohistochemistry suggests that peripherin and rhodopsin may follow different trafficking pathways to the ROS.
Keywords: 517 photoreceptors • 555 retina: distal(photoreceptors, horizontal cells, bipolar cells) • 606 transgenics/knock-outs