Isolation and Characterization of ROS and Disk Membranes
—ROS were isolated from mouse retinae. As demonstrated by transmission EM, scanning electron, and light microscopy, the protocol employed yielded highly enriched and structurally preserved ROS with a diameter of 0.85–1.4 μm and a length of 6–10 μm (). Thus, the diameters of ROS still attached to the retina () and isolated ROS (, b
) were comparable, suggesting structural integrity. Moreover, to check the quality of our preparations, we employed UV-visible spectroscopy and enzymatic assays of rhodopsin phosphorylation using intracellular endogenous rhodopsin kinase (18
) and reduction of the photoisomerized chromophore of rhodopsin (27
-retinal, using membrane impermeable [γ-32
P]ATP and [C4
H]NADPH, respectively (). The phosphorylation level was low (8.40 pmol) in untreated samples. Samples that were irradiated by light and subsequently sonicated expressed low quantities of phosphorylated rhodopsin as well. In contrast, the phosphorylation was the highest (49.40 pmol) in samples that were sonicated during light irradiation. The amount of [3
H]retinol was lowest in untreated samples and remained low in samples that were sonicated after light irradiation. In samples sonicated under light irradiation, the quantity of [3
H]retinol was the highest (). Thus, the results indicated that the isolated ROS were osmotically intact and that the rhodopsin molecules were fully active.
Fig. 1 Isolation and characterization of mouse ROS. a, scanning electron micrograph of mouse ROS attached to the retina. b, light micrograph of isolated ROS indicating the purity of the preparation. c, scanning electron micrograph of isolated ROS. d, transmission (more ...)
Disks isolated after osmotic bursting of the ROS and prepared by thin sectioning appeared as vesicles in the EM, compatible with the high osmotic pressure expected to inflate the structurally preserved disks (). Immunogold labeling of disks was performed using antibodies directed against the N-terminal (4D2 antibody) and C-terminal (1D4 antibody) ends of rhodopsin. More than 90% of the disks bound the C-terminal anti-rhodopsin antibody throughout the disk surface (, arrows). Less than 10% were labeled around their rim (, inset 1), suggesting that disrupted disks expose their extracellular surface. In agreement, about 10% of the disks were labeled when using the antibody directed against the rhodopsin N terminus (, inset 2). Taken together, the antibody labeling experiments strongly support the structural preservation of the disk membranes during their isolation. SDS-PAGE () revealed that the disk preparation did not contain significant amounts of soluble proteins normally present in ROS and was enriched in rhodopsin (>95%). The latter finding was identified by immunoblotting using the 4D2 and C7 antibodies (data not shown).
AFM Imaging of Rhodopsin in Native Disk Membranes
—To unveil the native supramolecular arrangement of rhodopsin, isolated disk membranes were adsorbed to freshly cleaved mica and imaged by AFM in buffer solution. The AFM was equipped with an infrared laser to avoid the formation of opsin, the retinal-depleted form of rhodopsin (28
). The morphology of an intact native disk adsorbed to mica is revealed in . Three different surface types are evident: the cytoplasmic side of the disk (type 1
), co-isolated lipid (type 2
), and mica (type 3
). Bare lipid bilayers had a thickness of 3.7 ± 0.2 nm (n
= 86) and an unstructured topography (, type 2
). Compared with the topography of the lipid, the cytoplasmic surface (type 1
) of the disk was highly corrugated, indicating the presence of densely packed proteins (see deflection image in ). Well adsorbed, single- and double-layered disk membranes had a thickness of 7–8 nm and 16–17 nm, respectively, a circular shape, and diameters between 0.9 and 1.5 μm. These disk diameters, determined by AFM, are in excellent agreement with those obtained from ROS by scanning electron microscopy (, a
) and light and electron microscopy (, b
). Open, spread-flattened disks adsorbed as round-shaped single-layered membranes to mica and exhibited four different surface types (). The first surface type (, type 1
) was characterized by a highly textured topography consisting of densely packed double rows of protrusions forming paracrystals (). SDS-PAGE revealed that rhodopsin was present at a high concentration in such disk membrane preparations (), suggesting that the visualized densely packed rows and paracrystals are related to this major protein. The second and third surface types were the same as in , i.e.
lipid and mica. The fourth surface type (, type 4
, and ) had the same morphology as the first except that the paracrystals formed rafts of rhodopsin separated by lipid. At higher magnification, rhodopsin dimers from densely packed regions (, broken ellipses
) or raft-like cluster (, broken ellipses
) to break off the rows were seen, identifying them as the building blocks of the paracrystals. Occasionally, single rhodopsin monomers (, arrowhead
) were detected on such topographs. The packing density in surface type 1 areas ranged between 30,000 and 55,000 rhodopsin monomers/μm2
), similar to the packing density within the rhodopsin islands in surface type 4 areas (in the packing density is about 34,000 rhodopsin monomers/μm2
). Obviously, the overall packing density of rhodopsin measured by AFM on tightly packed regions () or within rhodopsin rafts () is higher than that measured by optical methods (29
Fig. 2 Morphology of intact native disks adsorbed to mica and imaged in buffer solution. Shown are height (a) and deflection (b) images of an intact disk membrane having a typical thickness of 16–17 nm. Three different surface types are evident: the (more ...)
Fig. 3 Topography of an open, spread-flattened disk adsorbed to mica and imaged in buffer solution. a, height image of the open, spread-flattened disk. Four different surface types are evident: the cytoplasmic surface of the disk (types 1 and 4), lipid (type (more ...) AFM Imaging of Opsin in Native Disk Membranes
—The 65-kDa protein RPE65 is highly expressed in RPE cells and is one of the proteins involved in retinoid processing (reviewed in Ref. 28
). In Rpe65
−/− mice, retinoid analyses revealed no detectable 11-cis
-products in any of the ester, aldehyde, or alcohol forms (16
). Although these mice are able to develop ROS, the ROS contain opsin instead of rhodopsin. We used preparations of disks from Rpe65
−/− mice to compare the structure and the native supramolecular arrangement of opsin with that of rhodopsin.
In general, the morphology of the Rpe65
−/− disk membranes was similar to that of the wild-type membranes (see ), but occasionally, even better ordered paracrystals could be found in Rpe65
−/− preparations (). From such areas, power spectra (, inset
) were calculated and the unit cell parameters determined (a
= 8.4 ± 0.3 nm, b
= 3.8 ± 0.2 nm, γ = 85 ± 2° (n
= 9)), these values being the same as those found for wild-type paracrystals (15
). At higher magnification, rows of opsin dimers forming the paracrystal (, broken ellipse
) were visualized, indicating the same oligomeric state as rhodopsin in its native environment (compare with , b
, and with Ref. 15
). Occasionally, single-opsin monomers (, arrowheads
) were seen in such topographs. As with rhodopsin (15
), opsin protruded by 1.4 ± 0.2 nm (n
= 32) out of the lipid moiety on the cytoplasmic surface.
On the extracellular surface, no opsin paracrystals were evident (). The surface was corrugated, irregular, and flexible, preventing the acquisition of highly resolved AFM topographs such as required to reveal the paracrystalline packing. Opsin clusters (, triangle
) protruded 2.8 ± 0.2 nm (n
= 60) out of the lipid bilayer (, asterisk
) on the extracellular side, which is twice the height of the cytoplasmic protrusions. The latter finding is also in line with the atomic structure of rhodopsin determined by x-ray crystallography (6
). Irregular and flexible surfaces are typical for glycosylated proteins and proteins with long, flexible termini or loops. This observation, along with the fact that opsin has a long N terminus and is glycosylated on the extracellular surface, strengthens the assignment of this surface as the extracellular side of disk membranes. Similar difficulties were encountered with the glycosylated aquaporin-1 and the His-tagged AqpZ proteins where oligosaccharides or long termini impeded the acquisition of highly resolved surface topographs by AFM (30
). Similar observations were also made for the membranes containing rhodopsin instead of opsin (data not shown).
EM of Native Disk Membranes—To exclude the possibility of rhodopsin paracrystal formation upon adsorption on mica, native disk membranes were adsorbed on carbon-coated electron microscopy grids, negatively stained, and investigated by EM (). Power spectra were calculated from different regions of the adsorbed disks. Both the power spectra from a circular region adsorbed directly to the carbon film (, left PS) and from another region lying on disk membranes (, right PS) indicated diffraction patterns documenting the crystallinity of the disks irrespective of the support.
Higher Order Organization of Rhodopsin in Native Membranes
—The topographic information from AFM suggests a different packing arrangement of native rhodopsin dimers than of dimers observed in the three-dimensional crystal (5
). The thickness of single-layered disk membranes, 7.8 nm (15
), is compatible with the long axis, 7.5 nm, of the rhodopsin envelope derived from the 2.8-Å x-ray structure (6
). This indicates that all rhodopsin molecules are integrated with their long axes perpendicular to the bilayer. The extracellular protrusion measured by AFM, 2.8 nm, is compatible with that estimated from the x-ray structure, 2.7 nm. However, the measured cytoplasmic protrusions of rhodopsin and of opsin, 1.4 nm, are significantly smaller than the 1.8 nm estimated from the atomic model.
Unit cell dimensions (a
= 8.4 nm, b
= 3.8 nm, γ = 85°) of native rhodopsin and opsin paracrystals impose stringent boundary conditions for the packing arrangement of the rhodopsin/opsin dimers. The corresponding surface area (31.8 nm2
) barely suffices to house two rhodopsin molecules whose cross-section fits in a rectangle of 4.8 × 3.7 nm2
). Thus, a small number of packing models emerged that were thoroughly tested for steric clashes and natures of contacts. The best model revealing the different intra- and interdimeric contacts is shown in . The largest area of contact is 578 Å2
and intuitively represents the strongest interaction between rhodopsin molecules. It is found between helices IV and V, indicating this as the intradimeric contact (, contact 1
). Contacts involving helices I and II and the cytoplasmic loop between helices V and VI exhibit an area of 333 Å2
(, contact 2
) and represent the intra-row contacts. Finally, rows are weakly held together by interactions between regions of helix I close to the extracellular surface (, contact 3
) with a contact area of 146 Å2
Fig. 6 Model for the packing arrangement of rhodopsin molecules within the paracrystalline arrays in native disk membranes. a, rhodopsin assembles into dimers through a contact provided by helices IV and V (contact 1). Dimers form rows (highlighted by a blue (more ...)
Interactions within the Rho1–Rho2 dimer structure are located on both the cytoplasmic and the extracellular side. In the cytoplasmic part, hydrogen bonds dominate. These interactions include (Rho1 Arg147)–(Rho2)Asn145 and, symmetrically, (Rho1)Asn145–(Rho2)Arg147, which are located in the cytoplasmic loop between helices III and IV (C-II). Steric clashes are observed neither between the flat walls formed by helices IV and V nor between the C-II loops. In the extracellular region, the two Asn199 located at the end of helix V are within an appropriate distance from each other to form a hydrogen bond (atomic coordinates were deposited with Protein Data Bank accession number 1N3M). There is also one strong hydrophobic interaction near this site between the two Trp175 residues located in the loop between helices IV and V (E-II) in Rho1 and Rho2.
Interactions between dimers are formed only on the cytoplasmic side. They are mainly hydrophilic with hydrogen bonds between Lys339 (C-terminal) and Gln236 (C-III) and between Thr340 (C-terminal) and Gln238 (C-III). There is also a potential ionic bond in the membrane between Glu150 (C-II) and Lys231 (C-III). Interestingly, a line of positive residues spanning rhodopsin molecules as well as another line of negative side chains running across the rhodopsin oligomer are observed at the cytoplasmic surface ().