Jillian N. Pearring

Protein sorting, targeting and trafficking in photoreceptor cells

Vision is the most fundamental of our senses initiated when photons are absorbed by the rod and cone photoreceptor neurons of the retina. At the distal end of each photoreceptor resides a light-sensing organelle, called the outer segment, which is a modified primary cilium highly enriched with proteins involved in visual signal transduction. At the proximal end, each photoreceptor has a synaptic terminal, which connects this cell to the downstream neurons for further processing of the visual information. Understanding the mechanisms involved in creating and maintaining functional compartmentalization of photoreceptor cells remains among the most fascinating topics in ocular cell biology. This review will discuss how photoreceptor compartmentalization is supported by protein sorting, targeting and trafficking, with an emphasis on the best-studied cases of outer segment-resident proteins.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsins activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Guanylate cyclase 1 relies on rhodopsin for intracellular stability and ciliary trafficking

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin’s role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein. DOI: http://dx.doi.org/10.7554/eLife.12058.001

Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release

The primary cilium is a highly conserved organelle housing specialized molecules responsible for receiving and processing extracellular signals. A recently discovered property shared across many cilia is the ability to release small vesicles called ectosomes, which are used for exchanging protein and genetic material among cells. In this study, we report a novel role for ciliary ectosomes in building the elaborate photoreceptor outer segment filled with hundreds of tightly packed “disc” membranes. We demonstrate that the photoreceptor cilium has an innate ability to release massive amounts of ectosomes. However, this process is suppressed by the disc-specific protein peripherin, which enables retained ectosomes to be morphed into discs. This new function of peripherin is performed independently from its well-established role in maintaining the high curvature of disc edges, and each function is fulfilled by a separate part of peripherin’s molecule. Our findings explain how the outer segment structure evolved from the primary cilium to provide photoreceptor cells with vast membrane surfaces for efficient light capture.

R9AP targeting to rod outer segments is independent of rhodopsin and is guided by the SNARE homology domain

R9AP, the membrane anchor for transducins GTPase-activating complex, contains targeting information within its SNARE homology domain that is both necessary and sufficient for R9AP delivery to photoreceptor outer segments. R9APs targeting is independent of rhodopsin, the most abundant protein residing in the outer segment organelle.

Loss of Arf4 causes severe degeneration of the exocrine pancreas but not cystic kidney disease or retinal degeneration

Arf4 is proposed to be a critical regulator of membrane protein trafficking in early secretory pathway. More recently, Arf4 was also implicated in regulating ciliary trafficking, however, this has not been comprehensively tested in vivo. To directly address Arf4’s role in ciliary transport, we deleted Arf4 specifically in either rod photoreceptor cells, kidney, or globally during the early postnatal period. Arf4 deletion in photoreceptors did not cause protein mislocalization or retinal degeneration, as expected if Arf4 played a role in protein transport to the ciliary outer segment. Likewise, Arf4 deletion in kidney did not cause cystic disease, as expected if Arf4 were involved in general ciliary trafficking. In contrast, global Arf4 deletion in the early postnatal period resulted in growth restriction, severe pancreatic degeneration and early death. These findings are consistent with Arf4 playing a critical role in endomembrane trafficking, particularly in the pancreas, but not in ciliary function.

Progressive Rod–Cone Degeneration (PRCD) Protein Requires N-Terminal S-Acylation and Rhodopsin Binding for Photoreceptor Outer Segment Localization and Maintaining Intracellular Stability

The light-sensing outer segments of photoreceptor cells harbor hundreds of flattened membranous discs containing the visual pigment, rhodopsin, and all the proteins necessary for visual signal transduction. PRCD (progressive rod-cone degeneration) protein is one of a few proteins residing specifically in photoreceptor discs, and the only one with completely unknown function. The importance of PRCD is highlighted by its mutations that cause photoreceptor degeneration and blindness in canine and human patients. Here we report that PRCD is S-acylated at its N-terminal cysteine and anchored to the cytosolic surface of disc membranes. We also showed that mutating the S-acylated cysteine to tyrosine, a common cause of blindness in dogs and a mutation found in affected human families, causes PRCD to be completely mislocalized from the photoreceptor outer segment. We next undertook a proteomic search for PRCD-interacting partners in disc membranes and found that it binds rhodopsin. This interaction was confirmed by reciprocal precipitation and co-chromatography experiments. We further demonstrated this interaction to be critically important for supporting the intracellular stability of PRCD, as the knockout of rhodopsin caused a drastic reduction in the photoreceptor content of PRCD. These data reveal the cause of photoreceptor disease in PRCD mutant dogs and implicate rhodopsin to be involved in PRCDs unknown yet essential function in photoreceptors.