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

Abstract

Retinal degeneration slow (Rds) is a photoreceptor-specific tetraspanin glycoprotein essential for photoreceptor outer segment (OS) morphogenesis. Over 80 mutations in this protein are associated with several different retinal diseases. Rds forms a mixture of disulfide-linked homomeric dimers, octamers, and higher-order oligomers, with Cys150 playing a crucial role in its oligomerization. Rds also forms noncovalent homo-and hetero-tetramers with its nonglycosylated homologue, Rom-1. Here, we evaluated the subcellular site of Rds oligomerization and the pattern of Rds/Rom-1 complex assembly in several types of knockout mice, including rhodopsin (Rho −/− , lacking rod OS), Rom-1 (Rom-1 −/− ), neural retina leucine zipper (Nrl −/− , cone-dominant), and in comparison with wild-type (WT, rod-dominant) mice. Oligomerization and the pattern of complex assembly were also evaluated in OS-enriched vs OS-depleted preparations from WT and Rom-1 −/− retinas. Velocity sedimentation under reducing-and nonreducing conditions and co-immunoprecipitation experiments showed the presence of Rds mainly as homo-and hetero-tetramers with Rom-1 in the photoreceptor inner segment (IS), while higher-order, disulfide-linked intermediate complexes and oligomers were exclusively present in the photoreceptor OS. Rom-1-independent oligomerization of Rds was observed in Rom-1 −/− retinas. The pattern of Rds complexes in cones from Nrl −/− mice was comparable to that in rods from WT mice. On the basis of these findings, we propose that Rds traffics from the IS to the OS as homo-and hetero-tetramers, with subsequent disulfide-linked oligomerization occurring concomitant with OS disc morphogenesis (at either the base of OS or the tip of the connecting cilium). These results suggest that Rds mutations that interfere with tetramer formation can block Rds trafficking to the OS, leading to loss-of-function defects.

Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

“…Ablation of C150 in C150S/rds 2/2 transgenic mice eliminates the formation of disulfide-bonded complexes and as a consequence, these mice lack OSs, demonstrating that higher order oligomers are crucial for disc rim formation in rods and cones (13,20). It is not clear what the precise role of ROM1 is in this process, but we have observed that ROM1 is only found in the tetrameric and intermediate complexes but not in the larger oligomers (15).…”

“…Non-reducing velocity sedimentation was performed on whole retinal extract as described previously (13,15). Continuous density gradients of 5 -20% sucrose were prepared by sequentially layering 0.5 ml each of PBS-buffered 20, 15, 10 and 5% sucrose, containing 0.1% Triton X-100 and 10 mM N-ethylmaleimide (Sigma-Aldrich).…”

“…The samples were then analyzed by SDS -PAGE and western blotting. Molecular weight markers for the determination of tetrameric, octomeric and oligomeric fractions were used and have been published previously (15,24). Summary graphs for velocity sedimentation experiments were prepared by summing the percent of total RDS found in each fraction corresponding to the relevant complex type-oligomeric (fractions 1-3), intermediate (fractions 4 -5) and tetrameric (fractions 6 -9).…”

“…The methods employed for tissue collection and processing for plastic-embedment light and electron microscopy and immunogold labeling were as described previously (3,10,15). For light microscopy, 0.75 mm sections were viewed and photographed with an Olympus BH-2 photomicroscope with a Nikon digital camera system.…”

“…All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org segment (IS), RDS forms non-covalently bound homo-and hetero-tetramers with its homolog ROM1 (3,10,(15)(16)(17), another tetraspanin protein with similar localization and structure to RDS (18,19). After trafficking to the OS, homo-and hetero-tetrameric RDS complexes are linked to octomers while RDS homo-tetramers are linked to higher order oligomers through C150-mediated disulfide bonds.…”