AbMole Mepiroxol peripherin is a member of the tetraspanin family with the characteristic topology of four transmembrane domains, a large extracellular/intradiscal loop, and relatively short cytoplasmic N and C-termini. Peripherin localizes specifically to the rims of outer segment disc membranes and plays a crucial role in outer segment morphogenesis. This role is particularly highlighted in rds mice, in which the peripherin gene is severely truncated, essentially making them a peripherin knockout. These mice completely lack photoreceptor outer segments and instead display rudimentary stumps lacking disc structures. Consistent with mouse studies showing a requirement for peripherin in outer segment morphogenesis, over 90 different mutations in human peripherin have been associated with visual impairments. Unlike rhodopsin, it is unclear how peripherin is delivered to the outer segment. One of the first studies to examine this question showed that peripherin accumulates in intracellular vesicles while rhodopsin accumulates in the plasma membrane of photoreceptors in detached cat retinas. Results obtained in dying photoreceptors are difficult to interpret, but this finding may be viewed as indirect evidence that under normal conditions peripherin and rhodopsin utilize separate transport pathways. No mislocalized peripherin was found in any mouse models in which rhodopsin is knocked out or mislocalized, thus establishing that peripherin can be delivered independently of rhodopsin. However, this does not preclude peripherin from travelling in the same vesicles as rhodopsin under normal conditions. Studies examining photoreceptor targeting of C-terminal fragments of peripherin fused to a GFP reporter construct revealed that an amino acid stretch is necessary to target a reporter to Xenopus rod outer segments. Notably, this twenty amino acid sequence overlaps with a functional domain of peripherin implicated in membrane fusion. The essential requirement for peripherin in outer segment morphogenesis prompted us to further characterize its outer segment targeting. Targeting signals are often 4�C7 amino acids long, with only 2�C3 residues being critical to the specific targeting of the protein. Our goals were to narrow the previously identified peripherin targeting sequence, determine whether the targeting and the fusogenic domains were separable, and identify its most critical residues. Here we show that the targeting sequence is confined within ten amino acid residues, which do not overlap with the reported fusogenic domain, and that only a single amino acid within this region is irreplaceable �C a highly conserved valine at position 332. This construct retains two palmitoylated cysteine residues critical for membrane attachment, but lacks specific outer segment targeting information. When expressed in Xenopus under the rhodopsin promoter, the majority of the reporter localizes to the outer segment, but a distinct portion spills into the photoreceptor plasma membrane domain. This pattern is consistent with the expression of other membrane proteins that lack specific targeting information and is most likely explained by the majority of the construct being non-specifically packaged into rhodopsin carrier vesicles, which are thought to comprise the majority of all transport vesicles in frog photoreceptors. The rest of the construct is likely to be nonspecifically packaged into transport vesicles carrying membrane proteins to other subcellular compartments.