Fig 2: Proposed function of ABCA4 in the endolysosomal membranes of RPE. (A) Normal RPE cell. 11cRAL released during proteolysis of rhodopsin within a phagolysosome condenses with PE on the luminal surface to form 11-cis-N-retinylidene-phosphatidylethanolamine (11c-N-ret-PE), which undergoes isomerization to form a mixture of all-trans (at) and 11c-N-ret-PEs. Both N-ret-PE isomers are flipped by ABCA4 to the cytoplasmic surface, where hydrolysis of N-ret-PE is driven by mass action through binding of 11cRAL by cellular retinaldehyde-binding protein (CRALBP) or the reduction of atRAL to atROL by retinol dehydrogenase type 11 (RDH11). The atROL is processed by the RPE visual cycle through esterification by lecithin retinol acyltransferase (LRAT) to yield an all-trans-retinyl ester such as all-trans-retinyl palmitate (atRP), isomerization by RPE65 to yield 11-cis-retinol (11cROL), and oxidation by retinol dehydrogenase type 5 (RDH5) to yield 11cRAL, which binds to CRALBP. 11cRAL leaves the RPE cell to regenerate visual pigments in the adjacent photoreceptor OS. (B) Abca4−/− mutant RPE cell. The lack of ABCA4 in RPE endolysosomes of Abca4−/− mice or STGD1 patients causes delayed clearance of retinaldehydes and hence higher concentrations of both free retinaldehydes and N-ret-PE. This leads to secondary condensation of atRAL or 11cRAL with N-ret-PE to form bisretinoids.

Fig 3: Split NanoBiT proximity complementation assay. (A) Schematic showing the design of the system. Misfolded membrane protein (ABCA4; blue) is tagged at the cytoplasmic C-terminus with SmBiT part of NanoLuc (dark red) and the LgBiT subunit (light red) is targeted to the plasma membrane by the N-terminus of RP2 (magenta). When misfolded protein is retained in the ER the SmBiT (dark red) and LgBiT (light red) cannot complement (upper panel). Small molecule rescue of ABCA4 plasma membrane traffic enables the SmBiT–LgBiT system to produce luminescence through proximity induced complementation of the NanoLuc (lower panel). (B) WT-RHO fused with SmBiT localises to the plasma membrane. The staining was performed with antibody 4D2 without a permeabilization step, as the 4D2 epitope is located on the extracellular N-terminus the protein. (C) P23H fused to SmBiT staining is observed in a perinuclear and reticular pattern, consistent with the ER, following a detergent permeabilization step to reveal 4D2 immunoreactivity. (D) P23H-SmBiT is not detected with 4D2 using a no-permeabilization protocol. (E) The RP2-LgBiT localised to the plasma membrane, when stained with anti-LgBiT antibody in permeabilized cells. (F) Intracellular WT-ABCA4-SmBiT was detected with the ABCA4 3F4 ABCAM antibody (yellow) and WT-ABCA4-SmBiT localising to the plasma membrane was detected using an Abbexa antibody (magenta) 48 h post-transfection analysis. Scale bars = 10 μm. (G) 24 h post-transfection with RHO-SmBiT + RP2-LgBiT and P23H-SmBiT + RP2-LgBiT plasmids luminescence signal was measured. Raw luminescence values are shown. Error bars are ± SD. n = 3. two-tailed Student’s t-test for RHO vs. P23H. (H) Luminescence signal was analysed 48 h post-transfection with ABCA4 variants + RP2-LgBiT plasmids in live HEK293T cells. Mean of fold change relative to WT ± SD. n = 4 independent experiments. One-way ANOVA and post-hoc analysis comparisons were performed only against WT-ABCA4-SmBit + RP2-LgBit sample. Significant differences are displayed.

Fig 4: ABCA4 colocalizes with endolysosomal markers. (A) Representative merged confocal images of retina/RPE sections from 2-mo-old wild-type BALB/c (Upper) and albino Abca4−/− (Lower) mice reacted with antibodies to ABCA4 (red) and LAMP1 (green). Note that ABCA4 and LAMP1 colocalize in wild-type RPE but not in the OS. LAMP1, but not ABCA4, immunoreactivity is also present in the Abca4−/− RPE cells. (B) Representative merged confocal images of retina sections from 5-mo-old wild-type (129/Sv) (Top), Abca4−/− (Middle), and Mertk−/− (Bottom) mice immunostained with ABCA4 (red) and Rab5 (green) antibodies. Colocalization of ABCA4 and Rab5 is observed in both 129/Sv and Mertk−/− RPE cells as indicated by the orange signal. In the RPE of Abca4−/− retina section only Rab5 immunoreactivity is seen. White arrows indicate retinal detachment, and the white asterisk indicates the absence of OS in the Mertk−/− retina due to photoreceptor degeneration. (C) Representative confocal images of fixed hfRPE cells labeled with ABCA4 (red) (Top) or endosomal CAV1 (green) (Middle) antibodies. (Bottom) Merged confocal images of ABCA4 and CAV1. Note the colocalization of ABCA4 and CAV1. The green labeling of the filter in the CAV1 panel is due to nitrocellulose autofluorescence. Nuclei are stained with DAPI (blue). For murine RPE sections, n = 3 mice per group. For hfRPE cells, each experiment was repeated three times with three different donor cell lines. (Magnification: C, 60×.) (Scale bars, 10 μm.)

Fig 5: ABCA4 protein expression and VLP-targeting analysis.A, Western blot of ABCA4 protein expression in High Five (Hi5) insect cell lysates and VLP preparations using antibody against ABCA4. From left to right, lanes represent: Spectra Multicolor High Range Protein Ladder (Cat# 26625), ABCA4 from HEK293 lysate (positive control), Hi5 cell lysate infected with VLP-producing negative control baculovirus (negative control), ABCA4-VLP–producing Hi5 cell lysate, control VLP, and ABCA4-VLP. Two distinct bands were observed, with the top band likely indicative of posttranslational modifications occurring in ABCA4. Only the ABCA4-expressing cells and ABCA4-VLP samples show a specific ABCA4 signal. Samples were loaded based on an equal total protein concentration, as determined by the Bradford assay. B, Western blot analysis of the purified ABCA4-VLP fractions with size-exclusion chromatography (Izon qEV1/70 nm). The initial 2.8 ml was pooled as the default buffer volume (void volume). Fractions were loaded as 20 μl. VLP, virus-like particle.