Scheme of retinoid visual cycle. RDH, retinol dehydrogenase.

Isomerohydrolase activity in QBI-293A cells expressing RPE65 and LRAT. (A) Western blot analysis of total cell lysates. Lane 1, 10 μg of microsomal proteins from bovine RPE (for the RPE65 blot) and 1.25 μg of cell lysates of Sf9 cell expressing LRAT (for the LRAT blot) as positive controls; lanes 2–5, 50 μg of total proteins from 293A cells infected by Ad-GFP (lane 2), transfected with the LRAT plasmid alone (lane 3), infected with Ad-RPE65 alone (lane 4), and transfected with the LRAT plasmid and infected with Ad-RPE65 (lane 5). (B–E) Retinoids generated after incubation of 250 μg of total cellular protein with 0.2 μM all-trans [³H]retinol for 1.5 h. (B) Cells infected by Ad-GFP. (C) Cells infected with Ad-RPE65 alone. (D) Cells transfected with the LRAT plasmid alone. (E) Cells infected by Ad-RPE65 and transfected with LRAT. (F) 11-cis [³H]retinol standard. Peaks 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; 5, all-trans retinol.

Dependence of isomerohydrolase activity on RPE65 expression levels. (A) Time course of 11-cis retinol generation in 293A cell lysates expressing both LRAT and RPE65. (B) Time course of 11-cis retinol generation in bovine RPE homogenates. (C) Dependence of isomerohydrolase activity on MOI of Ad-RPE65. Lysates (250 μg) of 293A cells infected by Ad-RPE65 with increasing MOI and transfected with the same amount of the LRAT plasmid or 33 μg of bovine RPE homogenate were incubated with 0.2 μM all-trans [³H]retinol. In A–C, 11-cis [³H]retinol generated from the reaction were calculated from the area of the 11-cis retinol peak (mean ± SD, n = 3). (D) Western blot analysis of the same lysates used for the activity assay in C. PC, positive control, 10 μg of microsomal proteins from bovine RPE (for the RPE65 blot) and 1.25 μg of cell lysates of Sf9 cell expressing LRAT (for the LRAT blot); NC, negative control (40 μg of total protein from untreated cells); remaining lanes: 40 μg cell lysates from cells transfected with the same amount of LRAT plasmid and infected with Ad-RPE65 at different MOI as indicated. (E) Dependence of isomerohydrolase activity on the RPE65 expression level.

Isomerohydrolase activity in COS-1 cells expressing both RPE65 and LRAT. (A) Western blot analysis of COS-1 cells coexpressing LRAT and RPE65. Lanes 1, positive control 10 μg of microsomal proteins from bovine RPE (for the RPE65 blot) and 1.25 μg of cell lysates of Sf9 cells expressing LRAT (for the LRAT blot); 2, negative control (50 μg of total proteins from untreated cells); 3, 50 μg of cell lysate of COS-1 cells transfected with the LRAT expression plasmid and infected with Ad-RPE65 at MOI of 40; 4, 50μg of cell lysate transfected with the same amount of the LRAT plasmid and infected with Ad-RPE65 at MOI of 300. (B–F) Retinoids generated after incubation of 250 μg of COS-1 cell lysates with 0.2 μM all-trans [³H]retinol for 1.5 h: untreated cells (B), cells infected by Ad-RPE65 alone (C), cells transfected by the LRAT plasmid alone (D), cells transfected with the LRAT plasmid and infected by Ad-RPE65 at MOI of 40 (E), and cells transfected with the LRAT plasmid and infected by Ad-RPE65 at MOI 300 (F). Peaks 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; 5, all-trans retinol.

Colocalization of isomerohydrolase activity with RPE65 and LRAT. (A–C) Double staining of 293A cells expressing RPE65 and LRAT with antibodies specific for RPE65 (A, red) and LRAT (B, green) demonstrated an identical staining pattern of RPE65 and LRAT. (C) Superimposed image of A and B. QBI-293A (D–F) and COS-1 (G–I) cells expressing both RPE65 and LRAT were lysed and centrifuged at 100,000 × g for 1 h. The same amount of proteins (250 μg) from total protein before centrifugation (D and G), the supernatant (E and H), and pellet (F and I) after centrifugation was incubated with 0.2 μM all-trans [³H]retinol in the isomerohydrolase assay buffer for 1.5 h. Peaks were identified as follows: 1, retinyl esters; 2, all-trans retinal; 3, 11-cis retinol; 4, 13-cis retinol; 5, all-trans retinol. (J) Western blot analysis of 40 μg of protein from cell fractionations with antibody for RPE65. Lanes 1, total cell lysate; 2, supernatant from 293A cell after the centrifugation; 3, pellet from 293A cell after the centrifugation; 4, total cell lysate; 5, supernatant from COS-1 cells after the centrifugation; and 6, pellet from COS-1 cells after the centrifugation. MOI for Ad-RPE65 was 40 for 293A cells and 320 for COS-1 cells.

Proposed model for functional interactions between LRAT and RPE65. LRAT resides in the membrane via two transmembrane domains. RPE65 is associated with the membrane by three palmitoylated cysteines (indicated by C) (32). LRAT converts all-trans retinol to all-trans-retinyl ester, which is transported to RPE65 through the membrane. RPE65 obtains its substrate, highly hydrophobic all-trans-retinyl ester from the membrane and converted it into 11-cis retinol. at-ROL, all-trans retinol; at-RE, all-trans retinyl ester; 11-cis-ROL; 11-cis retinol.