SARA-Regulated Vesicular Targeting Underlies Formation of the Light-Sensing Organelle in Mammalian Rods

Abstract

The light-sensing organelle of the vertebrate rod photoreceptor, the outer segment (OS), is a modified cilium containing approximately 1,000 stacked disc membranes that are densely packed with visual pigment rhodopsin. The mammalian OS is renewed every ten days; new discs are assembled at the base of the OS by a poorly understood mechanism. Our results suggest that discs are formed and matured in a process that involves specific phospholipid-directed vesicular membrane targeting. Rhodopsin-laden vesicles in the OS axonemal cytoplasm fuse with nascent discs that are highly specialized with abundant phosphatidylinositol 3-phosphate (PI3P). This membrane coupling is regulated by the FYVE domain-containing protein, SARA, through its direct interaction with PI3P, rhodopsin, and SNARE protein syntaxin 3. Our model, in contrast to the previously proposed evagination model, suggests that the vesicular delivery of rhodopsin in the OS concentrates rhodopsin into discs, and this process directly participates in disc biogenesis.

Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet–Biedl syndrome

“…4). Consistent with the previous observations (33,34), Stx3 and Stxbp1 were found enriched near the connecting cilium, including areas (Fig. S5).…”

“…Stx3 and Stxbp1 are components of the SNARE complex that mediates membrane fusion events, which is the final step of vesicle trafficking. Indeed, Stx3 has been linked to Rho trafficking in photoreceptors (33,34). An insufficient supply of Stx3 and Stxbp1 in the IS may result in a delay or failure of vesicle fusion at the IS/OS interface.…”

Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

“…The SNAREs, Syntaxin 3 and Munc-18, have been localized to the IS, cilium, and OS, with syntaxin 3 localized primarily to the OS base (6,19), and they have a role in the fusion of rhodopsin carriers with the plasma membrane of the IS (40). Syntaxin 3 has also been proposed to regulate vesicle fusion with the forming discs at the base of the OS in a complex with rhodopsin and SARA (6). However, we found that whereas syntaxin 3 and Munc-18 are on the IS plasma membrane apposing the forming OS discs, they are largely absent from the OS base.…”

“…The evagination model proposes that the ciliary plasma membrane evaginates to form discs that are initially exposed to the extracellular space and then pinch off to form fully closed discs (4). In contrast, the fusion model proposes that discs originate from rhodopsincontaining vesicles, which undergo homotypic fusion to form new discs (5,6). Conventional electron microscopic (EM) analyses of chemically fixed specimens have clearly shown discs open to the extracellular space at the base of the OS, supporting the evagination model (4,7,8).…”

Heterotrimeric Kinesin-2 (KIF3) Mediates Transition Zone and Axoneme Formation of Mouse Photoreceptors

“…A conditional KIF3A knock-out in mouse cones prevented trafficking of opsins and other phototransduction components (32). However, mouse rods deficient in KIF3A showed normal trafficking of phototransduction components even as rods underwent rapid degeneration (32), suggesting redundancy of rod anterograde IFT motor(s) or motor-independent trafficking pathways (33)(34)(35).…”