Purchase this article with an account.
Younghwa Shin, Olga Nikolaeva, Gennadiy P Moiseyev, Yusuke Takahashi, Jian-Xing Ma; A Mutant of RPE65 D477G Affects Subcellular Localization of Wild-type RPE65 by Formation of Protein Complexes. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6333.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RPE65 is the isomerohydrolase indispensable in the visual cycle. A number of point-mutations in RPE65 have been associated with autosomal recessive retinal diseases such as retinitis pigmentosa (RP) and Leber’s congenital amaurosis (LCA). D477G is the first dominant mutant of RPE65 reported. How D477G exerts its dominant-negative effect on wild-type RPE65 has yet to be investigated. This study aims to gain more insights into dominant negative mechanism of the D477G mutation of RPE65.
Human wild-type RPE65 (hRPE65) and its mutant D477G were fused with mCherry and eGFP, respectively. 293-LRAT cells were transfected with: 1) hRPE65-mCherry, 2) D477G-eGFP and 3) hRPE65-mCherry and D477G-eGFP. Their expression and subcellular localizations were analyzed by both Western blotting and fluorescence microscopy. For immunoprecipitation, 293-LRAT cells were co-transfected with: 1) hRPE65 with the 6His tag and D477G with the 1D4 tag, 2) hRPE65-6His and BCO-1-1D4, and 3) PPARα-6His and D477G-1D4. Co-immunoprecipitation analyses were performed to determine whether hRPE65 and D477G physically interact.
hRPE65-mCherry and D477G-eGFP displayed different subcellular localization patterns as shown by fluorescence microscopy. In co-expression of hRPE65-mCherry with D477G-eGFP, normal distribution of hRPE65 was disturbed. Co-immunoprecipitation assay showed that D477G-1D4 and hRPE65-6His have a physical interaction. The two negative controls, PPARα-6His and BCO-1-1D4, were not co-precipitated with either D477G-1D4 or hRPE65-6His.
Normal subcellular distribution of hRPE65 was affected by the presence of D477G, through physical interactions between hRPE65 and D477G. These results indicate that hRPE65 and D477G mutant likely form a complex which disturbs subcellular localization of hRPE65 and subsequently, diminishes its function.
This PDF is available to Subscribers Only