Abstract
Purpose:
Little is known about the molecular pathways used to traffic proteins to the photoreceptor outer segment and thus far only the molecular details for trafficking the visual pigment, rhodopsin, have been reported. To further understand the intracellular trafficking of other outer segment proteins, we examined the targeting of peripherin. Peripherin is an integral membrane protein localized to the edges of outer segment discs, where it plays an essential role in disc morphogenesis and maintenance. Peripherin has been previously reported to contain a 20 amino acid targeting sequence within its C-terminus that differs from the targeting sequence of rhodopsin. Our goals were to narrow the previously identified peripherin targeting sequence, determine whether peripherin’s targeting and previously described fusogenic regions were separable, and identify its most critical residues for targeting.
Methods:
Peripherin C-terminal peptides were fused to peripherally associated or integral membrane reporter constructs and expressed in rods of transgenic Xenopus laevis. The subcellular localization of these constructs was analyzed in transgenic tadpoles at developmental stage 45 using confocal microscopy. Full length peripherin was electroporated into mouse rods and its localization was analyzed 21 days post injection.
Results:
We found that a peptide containing only 10 amino acids of the peripherin targeting sequence was sufficient for directing either peripherally associated or integral membrane reporters exclusively to rod outer segments of transgenic tadpoles. Alanine scanning mutagenesis of individual residues within this 10 residue stretch revealed that only a single residue, V332, was critical for the accurate targeting of these reporter constructs to the outer segment. This conclusion was further verified by in vivo electroporation, where we found that expression of full-length Flag-tagged peripherin containing the V332A mutation completely abrogated its targeting in rods of peripherin knockout mice. Conversely, targeting was unaffected in wild type mice where mutant peripherin likely oligomerizes with endogenous wild type peripherin.
Conclusions:
The positioning of the peripherin targeting sequence was narrowed to 10 amino acids that do not overlap with peripherin’s previously described fusogenic region, and that only a single residue within this sequence is irreplaceable, V332.
Keywords: 648 photoreceptors •
689 retina: distal (photoreceptors, horizontal cells, bipolar cells)