Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

Molday, Robert S.; Molday, Laurie L.

doi:10.1083/jcb.105.6.2589

Cited by 173 publications

(93 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Isolation of Retina and ROS Membranes-Retina and ROS membranes from frozen bovine retinas were prepared as previously described (27,37,38). Highly enriched ROS disc membranes were prepared by an immunogold density perturbation procedure (27).…”

Section: Methodsmentioning

confidence: 99%

“…Disc membranes, which comprise 95% of total ROS membranes, were separated from the ROS plasma membrane by an immunogold density perturbation method (27,37). Western blots of crude retinal membranes, isolated ROS membranes, and disc membranes were probed for Atp8a2 for comparison with ABCA4, a marker for the disc membrane, and the cyclic nucleotide-gated channel subunit ␣-subunit CNGA1, a marker for the plasma membrane of ROS (33,44).…”

Section: Atp8a2 Mrna Expression In the Retinal Photoreceptor Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

Coleman¹,

Kwok

Molday

2009

Journal of Biological Chemistry

174

227

View full text Add to dashboard Cite

P 4 -ATPases comprise a relatively new subfamily of P-type ATPases implicated in the energy-dependent translocation of aminophospholipids across cell membranes. In this study, we report on the localization and functional properties of Atp8a2, a member of the P 4 -ATPase subfamily that has not been studied previously. Reverse transcription-PCR revealed high expression of atp8a2 mRNA in the retina and testis. Within the retina, immunofluorescence microscopy and subcellular fractionation studies localized Atp8a2 to outer segment disc membranes of rod and cone photoreceptor cells. Atp8a2 purified from photoreceptor outer segments by immunoaffinity chromatography exhibited ATPase activity that was stimulated by phosphatidylserine and to a lesser degree phosphatidylethanolamine but not by phosphatidylcholine or other membrane lipids. Purified Atp8a2 was reconstituted into liposomes containing fluorescent-labeled phosphatidylserine to measure the ability of Atp8a2 to flip phosphatidylserine across the lipid bilayer. Fluorescence measurements showed that Atp8a2 flipped fluorescent-labeled phosphatidylserine from the inner leaflet of liposomes (equivalent to the exocytoplasmic leaflet of cell membranes) to the outer leaflet (equivalent to cytoplasmic leaflet) in an ATP-dependent manner. Our studies provide the first direct biochemical evidence that purified P 4 -ATPases can translocate aminophospholipids across membranes and further implicates Atp8a2 in the generation and maintenance of phosphatidylserine asymmetry in photoreceptor disc membranes.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Atp8a2 Mrna Expression In the Retinal Photoreceptor Cellsmentioning

confidence: 99%

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

Coleman¹,

Kwok

Molday

2009

Journal of Biological Chemistry

174

227

View full text Add to dashboard Cite

show abstract

“…Preparation and Trypsin Digestion of ROS-ROS were isolated from either frozen bovine retina or human retina from donor eyes (University of British Columbia Eye Bank) using continuous sucrose gradient centrifugation as previously described (29). ROS (1 mg/ml protein) were lysed in hypotonic buffer (10 mM Tris, pH 7.4, 1 mM EDTA), pelleted by centrifugation, and resuspended in 0.25 ml of Tris buffer (20 mM Tris, pH 8.0, 0.5 mM EDTA).…”

Section: Methodsmentioning

confidence: 99%

Membrane Topology of the ATP Binding Cassette Transporter ABCR and Its Relationship to ABC1 and Related ABCA Transporters

Bungert¹,

Molday²,

Molday

2001

Journal of Biological Chemistry

155

130

View full text Add to dashboard Cite

ABCR is a member of the ABCA subclass of ATP binding cassette transporters that is responsible for Stargardt macular disease and implicated in retinal transport across photoreceptor disc membranes. It consists of a single polypeptide chain arranged in two tandem halves, each having a multi-spanning membrane domain followed by a nucleotide binding domain. To delineate between several proposed membrane topological models, we have identified the exocytoplasmic (extracellular/lumen) N-linked glycosylation sites on ABCR. Using trypsin digestion, site-directed mutagenesis, concanavalin A binding, and endoglycosidase digestion, we show that ABCR contains eight glycosylation sites. Four sites reside in a 600-amino acid exocytoplasmic domain of the N-terminal half between the first transmembrane segment H1 and the first multi-spanning membrane domain, and four sites are in a 275-amino acid domain of the C half between transmembrane segment H7 and the second multi-spanning membrane domain. This leads to a model in which each half has a transmembrane segment followed by a large exocytoplasmic domain, a multi-spanning membrane domain, and a nucleotide binding domain. Other ABCA transporters, including ABC1 linked to Tangier disease, are proposed to have a similar membrane topology based on sequence similarity to ABCR. Studies also suggest that the N and C halves of ABCR are linked through disulfide bonds.ABCR, formerly known as the rim protein, is an ATP binding cassette (ABC) 1 transporter found in vertebrate retinal photoreceptor cells (1-3). It is localized along the rim region of photoreceptor rod outer segment disc membranes (1, 4, 5) and more recently has been found in human foveal and peripheral cone outer segments (6). Several studies have implicated ABCR in the retinoid cycle, possibly functioning as a retinal extruder or retinal-phosphatidylethanolamine flippase to facilitate the removal of all-trans-retinal from disc membranes following the photobleaching of rhodopsin (7-9).Mutations in the ABCA4 gene encoding ABCR have been linked to Stargardt disease, a relatively common juvenile macular dystrophy characterized by a decrease in visual acuity, progressive bilateral atrophy of the central retina, and the accumulation of yellow deposits within the retinal pigment epithelial cell layer (2, 10 -13). Mutations in ABCA4 have also been linked to related disease variants, including late-onset fundus flavimaculatus (14), cone-rod dystrophy (15), retinitis pigmentosa-like dystrophy (16, 17), and age-related macular degeneration (18), although the latter remains controversial (19).ABCR is a member of the ABCA subclass of ABC transporters (20). Like most other mammalian ABC transporters, members of this subclass consist of a single long polypeptide chain organized into two tandemly arranged halves. Each half contains a membrane-spanning domain (MSD) followed by a cytoplasmic nucleotide binding domain (NBD). The ABCA subclass is distinguished from other ABC transporter subclasses by the presence of a large domain between the...

show abstract

“…It is also a major protein of the ROS plasma membrane. In the plasma membrane, it constitutes approximately 40% of the total membrane protein [77]. However, the plasma membrane lipid composition is distinctly different from that of the disks membranes [37,58].…”

Section: Cholesterol and Rhodopsin Function In Ros Membranesmentioning

confidence: 99%

The role of cholesterol in rod outer segment membranes

Albert

Boesze‐Battaglia

2005

Progress in Lipid Research

112

View full text Add to dashboard Cite

The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evlauated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholestrol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthremore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting ...

show abstract

Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method.

Cited by 173 publications

References 37 publications

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

Localization, Purification, and Functional Reconstitution of the P4-ATPase Atp8a2, a Phosphatidylserine Flippase in Photoreceptor Disc Membranes

Membrane Topology of the ATP Binding Cassette Transporter ABCR and Its Relationship to ABC1 and Related ABCA Transporters

The role of cholesterol in rod outer segment membranes

Contact Info

Product

Resources

About