Molecular characterization of peripherin-2 and rom-1 mutants responsible for digenic retinitis pigmentosa

Peripherin-2 and Rom-1 are homologous tetraspanning membrane proteins that assemble into noncovalent tetramers and higher order disulfide-linked oligomers implicated in photoreceptor disc morphogenesis. Individuals who coinherit a L185P peripherin-2 mutation and a null or G113E rom-1 mutation are afflicted with retinitis pigmentosa, whereas individuals who inherit only one defective gene are normal. We examined the expression, subunit assembly, and disulfide-mediated oligomerization of L185P and L185A peripherin-2 and L188P Rom-1 by velocity sedimentation, co-immunoprecipitation, and cross-linking. These mutants formed noncovalent dimers under disulfide-reducing conditions but failed to assemble into core tetramers. Under nonreducing conditions, L185P dimers formed disulfide-linked tetramers but not higher order oligomers. L185P coassembled with wild-type peripherin-2 and Rom-1 to form tetramers and higher order disulfide-linked oligomers characteristic of the wild-type proteins. The G113E Rom-1 mutant expressed 20-fold lower than wild-type Rom-1, indicating that it behaves mechanistically as a null allele. We conclude that Leu(185) of peripherin-2 (Leu(188) of Rom-1) is critical for tetramer but not dimer formation and that the core tetramer has 2-fold symmetry. Peripherin-2-containing tetramers are required for higher order disulfide-linked oligomer formation. The level of these oligomers is critical for stable photoreceptor disc formation and the digenic retinitis pigmentosa disease phenotype.     

A novel tetraspanin fusion protein, peripherin-2, requires a region upstream of the fusion domain for activity

Peripherin-2 (also known as peripherin/rds), a photoreceptor specific tetraspanin protein, is required to maintain normal cell structure through its role in renewal processes requiring membrane fusion. It is the first tetraspanin fusogen and has been shown to directly mediate fusion between disk membranes and opposing membranes to maintain the highly ordered structure of rod outer segments. Localized to the C terminus of human, bovine, and murine peripherin-2 is an amphiphilic fusion peptide domain (residues 312-326) and a highly conserved region upstream of this domain that we hypothesize is essential for fusogenic function. Our previous studies indicated that substitution of a threonine for a proline at position 296 within this highly conserved region enhanced fusion activity. In this study we wanted to determine whether this proline is essential with the introduction of three additional substitutions of proline with alanine, leucine, and glutamic acid. Wild type, P296T, P296A, P296L, and P296E mutants of peripherin-2 were expressed as His6-tagged full-length proteins in Madin-Darby canine kidney (MDCK) cells. All of the proteins were localized to intracellular membranes and detected as 42-kDa monomers by Western blot analysis. The wild type, P296A, and P296L assembled into core tetramers; in contrast the P296T and P296E formed higher order oligomers. Fusogenic activity of full-length protein expressed in MDCK membranes and purified protein reconstituted in model membrane liposomes was determined using fluorescence quenching techniques. Fusion activity was decreased in the P296L, P296A, and P296E mutants both in endogenous MDCK membranes and in model liposomes. Collectively, these results suggest that the proline at position 296 is necessary for optimal function.     

SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1.     

The tetraspanin protein peripherin-2 forms a complex with melanoregulin, a putative membrane fusion regulator

Peripherin-2, the product of the rds gene, is a tetraspanin protein. In this study, we show that peripherin-2 forms a complex with melanoregulin (MREG), the product of the Mreg locus. Genetic studies suggest that MREG is involved in organelle biogenesis. In this study, we explore the role of this protein in processes associated with the formation of disk membranes, specialized organelles of photoreceptor rod cells. MREG antibodies were generated and found to be immunoreactive with a 28 kDa protein in retinal extracts, bovine OS, ARPE-19 cells, and rat RPE. MREG colocalized with peripherin-2 in WT (CB6F1/J) and in rds+/- retinas. Western blots of serial tangential sections confirmed the close association of these two proteins within the IS and basal outer segment of rods. Immunoprecipitation (IP) of OS extracts showed formation of a complex between MREG and peripherin-2-ROM-1 hetero-oligomers. This interaction was confirmed with pulldown analyses in which the GST-PerCter protein selectively pulled down His-MREG and His-MREG selectively pulled down PerCter. Biacore analysis using peptide inhibitors and per-2 truncation mutant studies allowed us to map the MREG binding site on per-2 to the last five residues of the C-terminus (Gln341-Gly346), and kinetic data predicted a KD of 80 nM for PerCter-MREG binding. Finally, the effect of MREG on photoreceptor specific membrane fusion was assayed using a disk-plasma membrane cell free assay. Preincubation of target membranes with MREG resulted in a dose-dependent inhibition of fusion with an IC50 in the submicromolar range. Collectively, these results suggest that this newly identified protein regulates peripherin-2 function.     

The cGMP-gated channel and related glutamic acid-rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes

The rod cGMP-gated channel is localized in the plasma membrane of rod photoreceptor outer segments, where it plays a central role in phototransduction. It consists of alpha- and beta-subunits that assemble into a heterotetrameric protein. Each subunit contains structural features characteristic of nucleotide-gated channels, including a cGMP-binding domain, multiple membrane-spanning segments, and a pore region. In addition, the beta-subunit has a large glutamic acid- and proline-rich region called GARP that is also expressed as two soluble protein variants. Using monoclonal antibodies in conjunction with immunoprecipitation, cross-linking, and electrophoretic techniques, we show that the cGMP-gated channel associates with the Na/Ca-K exchanger in the rod outer segment plasma membrane. This complex and soluble GARP proteins also interact with peripherin-2 oligomers in the rim region of outer segment disc membranes. These results suggest that channel/peripherin protein interactions mediated by the GARP part of the channel beta-subunit play a role in connecting the rim region of discs to the plasma membrane and in anchoring the channel.exchanger complex in the rod outer segment plasma membrane.     

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.     

Glycoproteins specific for the retinal rod outer segment plasma membrane

Two ricin-specific glycoproteins have been identified on neuraminidase-treated rod outer segment plasma membranes of bovine retinal photoreceptor cells. Ricin-gold-dextran particles were observed by electron microscopy to densely label the surface of neuraminidase-treated rod outer segments. Western blotting of proteins separated by SDS-gel electrophoresis indicated that two ricin-binding glycoproteins of Mr 230,000 and 110,000 are specific for the plasma membrane and are not found in disk membranes. These glycoproteins can serve as specific probes for the purification of the rod outer segment plasma membrane.     

Peripherin-2: an intracellular analogy to viral fusion proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.     

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning³¹P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.     

Rhodopsin is spatially heterogeneously distributed in rod outer segment disk membranes

The visual photoreception takes place in the retina, where specialized rod and cone photoreceptor cells are located. The rod outer segments contain a stack of 500-2,000 sealed membrane disks. Rhodopsin is the visual pigment located in rod outer segment disks, it is a member of the G-protein-coupled receptor (GPCR) superfamily, an important group of membrane proteins responsible for the majority of physiological responses to stimuli such as light, hormones, peptides, etc. Alongside rhodopsin, peripherin/Rom proteins located in the disk rims are thought to be responsible for disk morphology. Here we describe the supramolecular structure of rod outer segment disk membranes and the spatial organization of rhodopsin and peripherin/Rom molecules. Using atomic force microscopy operated in physiological buffer solution, we found that rhodopsin is loosely packed in the central region of the disks, in average about 26?000 molecules covering approximately one third of the disk surface. Peripherin/Rom proteins form dense assemblies in the rim region. A protein-free lipid bilayer girdle separates the rhodopsin and peripherin/Rom domains. The described supramolecular assembly of rhodospin, peripherin/Rom and lipids in native rod outer segment disks is consistent with the functional requirements of photoreception.     

Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration

The outer segments of vertebrate rod photoreceptor cells consist of an ordered stack of membrane disks, which, except for a few nascent disks at the base of the outer segment, is surrounded by a separate plasma membrane. Previous studies indicate that the protein, peripherin or peripherin/rds, is localized along the rim of mature disks of rod outer segments. A mutation in the gene for this protein has been reported to be responsible for retinal degeneration in the rds mouse. In the present study, we have shown by immunogold labeling of rat and ground squirrel retinas that peripherin/rds is present in the disk rims of cone outer segments as well as rod outer segments. Additionally, in the basal regions of rod and cone outer segments, where disk morphogenesis occurs, we have found that the distribution of peripherin/rds is restricted to a region that is adjacent to the cilium. Extension of its distribution from the cilium coincides with the formation of the disk rim. These results support the model of disk membrane morphogenesis that predicts rim formation to be a second stage of growth, after the first stage in which the ciliary plasma membrane evaginates to form open nascent disks. The results also indicate how the proteins of the outer segment plasma membrane and the disk membranes are sorted into their separate domains: different sets of proteins may be incorporated into membrane outgrowths during different growth stages of disk morphogenesis. Finally, the presence of peripherin/rds protein in both cone and rod outer segment disks, together with the phenotype of the rds mouse, which is characterized by the failure of both rod and cone outer segment formation, suggest that the same rds gene is expressed in both types of photoreceptor cells.     

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.     

Identification of an outer segment targeting signal in the COOH terminus of rhodopsin using transgenic Xenopus laevis

Mislocalization of the photopigment rhodopsin may be involved in the pathology of certain inherited retinal degenerative diseases. Here, we have elucidated rhodopsin's targeting signal which is responsible for its polarized distribution to the rod outer segment (ROS). Various green fluorescent protein (GFP)/rhodopsin COOH-terminal fusion proteins were expressed specifically in the major red rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The fusion proteins were targeted to membranes via lipid modifications (palmitoylation and myristoylation) as opposed to membrane spanning domains. Membrane association was found to be necessary but not sufficient for efficient ROS localization. A GFP fusion protein containing only the cytoplasmic COOH-terminal 44 amino acids of Xenopus rhodopsin localized exclusively to ROS membranes. Chimeras between rhodopsin and alpha adrenergic receptor COOH-terminal sequences further refined rhodopsin's ROS localization signal to its distal eight amino acids. Mutations/deletions of this region resulted in partial delocalization of the fusion proteins to rod inner segment (RIS) membranes. The targeting and transport of endogenous wild-type rhodopsin was unaffected by the presence of mislocalized GFP fusion proteins.     

Immunocytochemical localization of opsin in the cell membrane of developing rat retinal photoreceptors

Mature retinal rod photoreceptors sequester opsin in the disk and plasma membranes of the rod outer segment (ROS). Opsin is synthesized in the inner segment and is transferred to the outer segment along the connecting cilium that joins the two compartments. We have investigated early stages of retinal development during which the polarized distribution of opsin is established in the rod photoreceptor cell. Retinas were isolated from newborn rats, 3-21 d old, and incubated with affinity purified biotinyl-sheep anti-bovine opsin followed by avidin- ferritin. At early postnatal ages prior to the development of the ROS, opsin is labeled by antiopsin on the inner segment plasma membrane. At the fifth postnatal day, as ROS formation begins opsin was detected on the connecting cilium plasma membrane. However, the labeling density of the ciliary plasma membrane was not uniform: the proximal cilium was relatively unlabeled in comparison with the distal cilium and the ROS plasma membrane. In nearly mature rat retinas, opsin was no longer detected on the inner segment plasma membrane. A similar polarized distribution of opsin was also observed in adult human rod photoreceptor cells labeled with the same antibodies. These results suggest that some component(s) of the connecting cilium and its plasma membrane may participate in establishing and maintaining the polarized distribution of opsin.     

In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and Rho(P23H)-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ～0.1% compared to endogenous opsin. Rho(P23H)-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with Rho(P23H)-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The Rho(P23H)-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing Rho(P23H), suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.     

Glyceraldehyde-3-phosphate dehydrogenase is a major protein associated with the plasma membrane of retinal photoreceptor outer segments

A major 38-kDa protein associated with bovine rod outer segment plasma membranes, but not disk membranes, has been identified as glyceraldehyde-3-phosphate dehydrogenase on the basis of its N-terminal sequence and specific enzyme activity. This enzyme was extracted from lysed rod outer segments or isolated rod outer segment plasma membrane with 0.15 M NaCl and purified to homogeneity by affinity chromatography on a NAD(+)-agarose column. A specific activity of 90-100 units/mg of protein is within the range of activity obtained for glyceraldehyde-3-phosphate dehydrogenase isolated from other mammalian cells. Enzyme activity measurements indicate that this enzyme makes up approximately 2% of the total rod outer segment protein and over 11% of the plasma membrane protein. Protease digestion and binding studies on purified rod outer segment plasma and disk membranes suggest that glyceraldehyde-3-phosphate dehydrogenase reversibly interacts with a protease-sensitive plasma membrane-specific protein of rod outer segments. The finding that glyceraldehyde-3-phosphate dehydrogenase is present in large quantities in rod outer segments suggests that at least some of the energy required for the synthesis of ATP and GTP for phototransduction and other processes of the outer segment is derived from glycolysis which takes place within this organelle.     

Specific binding of RGS9-Gbeta 5L to protein anchor in photoreceptor membranes greatly enhances its catalytic activity

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.