ROM-1 potentiates photoreceptor specific membrane fusion processes

Abstract

Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/ rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin's nonglycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-P/rds1 complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes.

Deciphering the Structure and Function of Als2cr4 in the Mouse Retina

“…38 As is evident from these studies, the loss of structural proteins has devastating effects on photoreceptor morphology and leads to loss of photoreceptors. Because Als2cr4 shares structural similarities, has a comparable immunostaining pattern, 24,34 and is embedded in the discs, we hypothesize that, similar to peripherin/rds and ROM-1, this structural protein contributes to maintaining the structural integrity of photoreceptors.…”

“…31 In retinal disease, the most prominent members of the tetraspanin family are peripherin/rds 32,33 and ROM-1. 34 These proteins participate in outer segment morphogenesis by providing a structural basis whereby new discs and lamellae are continually added to the photoreceptor outer segment basal region. 32 The outer segment renewal is a critical process as photoreceptors undergo daily phagocytosis of apical discs and lamellae by retinal pigment epithelium.…”

PRPH2/RDS and ROM-1: Historical context, current views and future considerations

“…Support for the hypothesis that PRPH2/RDS complex formation is important for membrane fusion came from experiments showing that various disruptions in the D2 loop and blocking of PRPH2/RDS disulfide mediated oligomerization disrupted the fusogenic activity of PRPH2/RDS (Boesze-Battagliaa and Stefano, 2002). In subsequent experiments using purified PRPH2/RDS and ROM-1 expressed in COS-1 cells, it was determined that ROM-1 could not promote membrane fusion alone but did enhance PRPH2/RDS fusogenic activity when in complex with PRPH2/RDS (Boesze-Battaglia et al, 2007b). These data led to a hypothesized model in which the PRPH2/RDS tetramers, intermediate and higher-order oligomers have variable abilities to initiate membrane fusion and together are involved directly in the complicated membrane dynamics at the base of the OS (Boesze-Battaglia et al, 1997).…”

Outer Segment Oligomerization of Rds: Evidence from Mouse Models and Subcellular Fractionation

“…Boesze-Battaglia and co-workers have demonstrated that Rds complexes are capable of mediating membrane fusion in vitro (34). More recently, it has been shown that Rom-1 is not capable of mediating membrane fusion alone but its presence in Rds/ Rom-1 complexes enhances Rds-mediated membrane fusion (35). The third known function of Rds in the OS is one of the focuses of this work.…”