Outer segment oligomerization of Rds: evidence from mouse models and subcellular fractionation.

Vertebrate rod and cone photoreceptors are highly complex neuronal cells that contain the components necessary for the initial events involved in the process of vision. These cells possess a structure called the “outer segment” (OS1), a unique organelle composed of hundreds of flattened membranous “discs”, the subcellular sites wherein the initial events of phototransduction take place. The inner segment (IS) functions as the cell body of the photoreceptor, and is linked to the OS by a connecting cilium. Retinal degeneration slow (Rds) and rod outer segment membrane protein 1 (Rom-1) are members of a tetra-spanning family present along the OS rim membranes (1, 2). Rds (also known as peripherin/Rds or peripherin-2) is required for proper photoreceptor OS morphogenesis and stabilization, since retinal degeneration slow (Rds−/−) mutant mice fail to develop OSs while heterozygous (Rds+/−) mice form highly disorganized OS structures consisting of dysmorphic whorls of membranes (3, 4). Moreover, Rds gene mutations have been found to associate with a variety of inherited human retinal diseases, including autosomal dominant retinitis pigmentosa (ADRP) and multiple classes of macular degeneration (5, 6). The C-terminus of Rds has been shown to promote membrane fusion in vitro, signifying a possible role in OS renewal (7), while the large intradiscal loop is known to play an important role in protein–protein interactions necessary for disc formation and stabilization (8–11). Rds also has been shown to associate with the rod cyclic nucleotide-gated (CNG) channel through the glutamic acid-rich protein (GARP) of the channel β subunit, which is thought to maintain the spatial arrangement of the disc so as to connect the disc rim region to the rod plasma membrane (12). Our recent work has shown that Rds plays different roles in cone versus rod photoreceptors (13, 14). Rod photoreceptors lacking Rds fail to form OS structures; hence, they exhibit minimal phototransduction activity and subsequently degenerate and die. In contrast, cone photoreceptors lacking Rds have markedly altered OS structure, with aberrant lamellar organization but are viable and capable of robust phototransduction (13). Rds shares many common structural features with its nonglycosylated counterpart, Rom-1 (2, 15). Both contain four transmembrane domains with short cytoplasmic N- and C-terminal regions and a large intradiscal loop of approximately 150 amino acids harboring seven highly conserved cysteine residues (2, 15, 16). Both in vivo and in vitro studies have demonstrated that noncovalent interactions between Rds and Rom-1 act to support the formation of homo- and hetero-tetrameric functional complexes (8, 17). Although the proper assembly of Rds and Rom-1 complexes is believed to play a crucial role in promoting and preserving normal OS structure, the functional activities and the sites of interaction between the two proteins at the molecular level are not completely understood. It is believed that the hetero-and homo-tetrameric complexes are linked together through intermolecular disulfide bonds to form octamers and higher-order oligomers that are crucial for disc rim formation (18). This oligomerization has been suggested to be mediated by a cysteine residue at position 150 (Cys150) in the large intradiscal loop (9, 18). In contrast to Rds, Rom-1 appears to play a lesser role in disc morphogenesis and structural maintenance, since Rom-1 knockout (Rom-1−/−) mice develop OSs whose morphology is only minimally or moderately abnormal (19). Additionally, unlike Rds, Rom-1 is not present in lower vertebrates (20–22), and previous work has demonstrated that Rds is essential for targeting Rom-1 to the OS (23). Furthermore, there is a direct correlation between Rds levels and the structural and functional properties of photoreceptor OSs (24). The present study was designed to elucidate the intracellular compartment of the photoreceptor cell wherein Rds oligomerization occurs. In particular, we examined more directly the role of disulfide-mediated oligomerization of Rds in the process of OS disc morphogenesis. Using a variety of well-characterized mouse strains, we show that Rds oligo-merization occurs in the OS of photoreceptors, that Rds can build higher-order complexes with or without Rom-1, and that Rds homo- and hetero-tetramers are formed mainly in the IS of photoreceptors. This is the first in vivo study demonstrating OS oligomerization of Rds and IS trafficking of Rds and Rom-1 core tetrameric complexes.