Purchase this article with an account.
Songhua Li, John Green, Jean Jacob, Minghao Jin; N-Terminal Region of Fatty Acid Transport Protein 4 (FATP4) is Important for Interacting with and Inhibiting RPE65 Isomerase. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3759.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In a previous study, we identified FATP4 as negative regulator of RPE65 isomerase, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis retinol (11cROL) for the visual cycle in the retinal pigment epithelium (RPE). The purpose of this study is to identify the region(s) of FATP4 that interacts with RPE65 and inhibits synthesis of 11-cis retinol.
A mammalian expression vector encoding the bovine FATP4 (pRK-FATP4) was used to construct various mutant FATP4s with a Flag tag. A series of N-terminal or C-terminal deletion mutations and the Ichthyosis Prematurity Syndrome (IPS)-associated missense or non-sense mutations (A92T, S247P, Q300R, and C168X) were individually introduced into pRK-FATP4. These mutant FATP4s and RPE65 were expressed in 293T cells stably expressing LRAT and/or CRALBP (293T-LC or 293T-C cells). Interaction of wild-type and mutant FATP4s with RPE65 in the cells was determined by immunoprecipitation. The inhibitory effects of the mutant FATPs on RPE65 activity were compared by measuring synthesis of 11cROL from all-trans retinyl palmitate or all-trans retinol added into the cell homogenates or medium of the culture cells.
Synthesis of 11-cis retinol in the cells transfected with pRK-RPE65 and pRK-FATP4 (wild-type FATP4) was 60% lower than that of control cells transfected with pRK-RPE65 and pRK5 mock vector. Under the same experimental conditions, C-terminal regions of FATP4 had no inhibitory effect on the synthesis of 11-cis retinol. These regions were not able to co-precipitate RPE65 in immunoprecipitation. In contrast, N-terminal region of FATP4 significantly inhibited synthesis of 11cROL. The inhibitory effect of the N-terminal region was appromaximitely 70% of wild-type FATP4. The N-terminal region of FATP4 was able to co-precipitate RPE65 in immunoprecipitation. The IPS-associated FATP4s, which lost very long-chain fatty acid-CoA synthetase activity, also inhibited synthesis of 11cROL. However, their inhibitory efficiencies were reduced 20-30% compared to the inhibitory efficiency of wild-type FATP4.
Our results indicate that the very long-chain fatty acid-CoA synthetase activity of FATP4 partially contributes to its inhibitory effect on synthesis of 11cROL and the N-terminal region of FATP4 is important for interacting with RPE65 and for inhibiting synthesis of 11cROL.
This PDF is available to Subscribers Only