January 1, 2013;
A single valine residue plays an essential role in peripherin/rds targeting to photoreceptor outer segments.
/retinal degeneration slow (rds
) is an integral membrane protein specifically localized to the light-sensing organelle of the photoreceptor cell, the outer segment. Within the outer segment, peripherin
is found at the edges of photoreceptor discs, where it plays a critical role in disc morphogenesis and maintenance. Peripherin
loss or mutations are often associated with severe forms of visual impairments. Like all other resident outer segment proteins, peripherin
is synthesized in the photoreceptor cell body and subsequently transported to the outer segment. In an effort to further examine peripherin''s delivery to outer segments, we undertook a careful examination of its targeting sequence. Using a fluorescently labeled reporter expressed in the rods of transgenic tadpoles, we narrowed peripherin''s targeting sequence to ten amino acids within its C-terminal tail
. This small stretch of amino acid residues is both necessary and sufficient for outer segment targeting. We also conducted alanine scanning of all residues within this sequence and found that only a single residue, valine at position 332, is essential for outer segment targeting. This valine is conserved in all species and its mutation is sufficient to completely abrogate the targeting of full-length peripherin
in mouse rods.
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Figure 2. Alanine scanning mutagenesis of the peripherin targeting sequence identifies V332 as the only residue essential for targeting.Panels A–I show confocal images of transgenic frog retinas expressing the YFP-xRhoCTδ5-xPer317–336 (green) variations containing single alanine substitutions of individual residues. (J) Multiple sequence alignment of peripherin targeting sequences from different species; residues with over 90% identity are highlighted yellow and the 100% conserved valine is highlighted orange. Abbreviations are: OS – outer segment, IS – inner segment, N – nuclei, ST – synaptic termini. Nuclei (blue) are stained with Hoechst. Scale bar 5 µm.
Figure 3. The peripherin targeting sequence redirects the subcellular targeting of the Htr1a serotonin receptor.Panels show confocal images of transgenic frog retinas expressing YFP-fused Htr1a constructs (green) schematically illustrated above each panel. (A) Htr1a-YFP; fluorescent signal observed in calycal processes is marked by white arrows. (B) Htr1a-YFP containing the Per327–336 sequence fused at the C-terminus. (C) Same construct as in (B), but bearing the V332A mutation. Abbreviations are: OS – outer segment, IS – inner segment, N – nuclei, ST – synaptic termini. Nuclei (blue) are stained with Hoechst. Scale bar: 5 µm.
Figure 4. The V332A mutation abrogates peripherin’s outer segment targeting in rods of the rds mouse.(A) Wild type FLAG-tagged mouse peripherin (mPer) was electroporated into rods of the rds mouse and stained with anti-FLAG antibodies (green). (B) V332A FLAG-tagged peripherin electroporated into rods of the rds mouse; construct mislocalization to the inner segment and synaptic terminal is highlighted by white arrowheads. (C) V332A FLAG-tagged peripherin electroporated into the rods of wild type mice. The electroporated constructs are illustrated schematically above the corresponding panels with the position of the FLAG tag depicted in red. Note that the outer segment shapes in (A) and (C) are different due to ongoing photoreceptor degeneration in the rds mouse (A). Abbreviations are: OS – outer segment, IS – inner segment, N – nuclei, ST – synaptic termini. Nuclei (blue) are stained with Hoechst. Scale bar: 10 µm.
The peripherin targeting signal is contained within a ten amino acid residue stretch.
Panels show confocal images of transgenic frog retinas expressing the reporter construct YFP-xRhoCTδ5 (green) fused to the fragments of the peripherin C-terminus illustrated in cartoons above the corresponding panels. Partial mislocalization of several constructs from rod outer segments is marked by white arrowheads. (A) The YFP-xRhoCTδ5 reporter. (B) The reporter fused to xPer 317–336. (C) The reporter fused to xPer 317–327. (D) The reporter fused to xPer 327–336. (E–G) The reporter fused to xPer 317–336 sequences containing polyalanine amino acid substitutions indicated below the panels. Abbreviations are: OS – outer segment, IS – inner segment, N – nuclei, ST – synaptic termini. The nuclei (blue) are stained with Hoechst; scale bar: 5 µm. (H) A schematic of a frog rod photoreceptor illustrating its principle compartments.