Nerative diseases [1?]. Membrane proteins destined for the outer segment are synthesized

Nerative diseases [1?]. Membrane proteins destined for the outer segment are synthesized in the endoplasmic reticulum, transported through the Golgi, and then sorted at the trans-Golgi network into transport vesicles specifically directed to the outer segment. The fidelity of sorting is guided by targeting signals, which are short stretches of amino acid residues encoding protein localization information [4,5]. These targeting signals often I-BRD9 biological activity reside within a protein’s cytoplasmic domain and are deciphered by protein sorting complexes present at the trans-Golgi. Only two targeting signals responsible for directing membrane proteins to the outer segment have been reported thus far. One signal is VXPX, which is shared by rhodopsin, cone opsins, and the photoreceptorspecific retinol dehydrogenase [6?], as well as several otherproteins targeted to primary and sensory cilia in other cell types [10?2]. In photoreceptors, this signal interacts with a small GTPase Arf4, which defines rhodopsin packaging into transport vesicles for outer segment delivery [13,14]. The second known targeting sequence resides within the Cterminus of peripherin/retinal degeneration slow (also known as rds or peripherin-2, hereafter referred to as peripherin) [15]. KS-176 manufacturer 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 [16,17]. This role is particularly highlighted in rds mice, in which the peripherin gene is severely truncated, essentially making them a peripherin knockout [18]. These mice completely lack photoreceptor outer segments and instead display rudimentary stumps lacking disc structures [19,20]. 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 (http:// www.retina-international.org/files/sci-news/rdsmut.htm). 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 photoreceptorsA Single Valine Defines Peripherin Targetingin detached cat retinas [21]. 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 23977191 which rhodopsin is knocked out or mislocalized [22,23], 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 (residues 317?36) is necessary to target a reporter to Xenopus rod outer segments [15]. Notably, this twenty amino acid sequence overlaps with a functional domain of peripherin implicated in membrane fusion [24?7]. The essential requirement for peripherin in outer segment morphogenesis prompted us to further characterize its outer segment targeting.Nerative diseases [1?]. Membrane proteins destined for the outer segment are synthesized in the endoplasmic reticulum, transported through the Golgi, and then sorted at the trans-Golgi network into transport vesicles specifically directed to the outer segment. The fidelity of sorting is guided by targeting signals, which are short stretches of amino acid residues encoding protein localization information [4,5]. These targeting signals often reside within a protein’s cytoplasmic domain and are deciphered by protein sorting complexes present at the trans-Golgi. Only two targeting signals responsible for directing membrane proteins to the outer segment have been reported thus far. One signal is VXPX, which is shared by rhodopsin, cone opsins, and the photoreceptorspecific retinol dehydrogenase [6?], as well as several otherproteins targeted to primary and sensory cilia in other cell types [10?2]. In photoreceptors, this signal interacts with a small GTPase Arf4, which defines rhodopsin packaging into transport vesicles for outer segment delivery [13,14]. The second known targeting sequence resides within the Cterminus of peripherin/retinal degeneration slow (also known as rds or peripherin-2, hereafter referred to as peripherin) [15]. 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 [16,17]. This role is particularly highlighted in rds mice, in which the peripherin gene is severely truncated, essentially making them a peripherin knockout [18]. These mice completely lack photoreceptor outer segments and instead display rudimentary stumps lacking disc structures [19,20]. 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 (http:// www.retina-international.org/files/sci-news/rdsmut.htm). 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 photoreceptorsA Single Valine Defines Peripherin Targetingin detached cat retinas [21]. 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 23977191 which rhodopsin is knocked out or mislocalized [22,23], 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 (residues 317?36) is necessary to target a reporter to Xenopus rod outer segments [15]. Notably, this twenty amino acid sequence overlaps with a functional domain of peripherin implicated in membrane fusion [24?7]. The essential requirement for peripherin in outer segment morphogenesis prompted us to further characterize its outer segment targeting.