Localization of Adipocyte Differentiation-Related Protein (ADRP) and TIP47 in the Retinal Pigment Epithelium

Abstract

Purpose:: Lipid droplets (LDs) are ubiquitous cytoplasmic structures found in virtually all cells. Recently, a structure called retinosome, or retinyl ester storage compartment, was shown to harbor retinyl esters in the retinal pigment epithelium (RPE), and was proposed to be involved in the retinoid cycle in the retina. The retinosome was similar to LDs in harboring adipocyte differentiation-related protein (ADRP). We investigated the localization of ADRP in the RPE. We also examined the behavior of another LD protein, TIP47.

Methods:: Mouse RPE in vivo and ARPE-19 cells were labeled by antibodies to ADRP and TIP47 for immunofluorescence microscopy. LDs were stained by BODIPY493/503. Immunoelectron microscopy was done for ADRP in mouse RPE. cDNAs of various TIP47 mutants were transfected to ARPE-19 cells and localization of the expressed proteins was examined quantitatively. Endogenous TIP47 was knocked down by RNA interference (RNAi). Effects of the mutants' overexpression and the TIP47 RNAi on the retinoids were examined by HPLC.

Results:: TIP47 accumulated to BODIPY493/503-labeled round structures in the light-adapted mouse RPE and ARPE-19 cells loaded all-trans-retinol. Those structures were invariably labeled for ADRP. By immunoelectron microscopy, ADRP was localized to canonical LDs. The redistribution of TIP47 to LDs was abolished when either the amino terminal or the carboxyl terminal half of the molecule was deleted, but enhanced by a short deletion in the carboxyl terminus or by a replacement of critical hydrophobic residues by alanines. Knockdown of endogenous TIP47 did not affect the amount of retinyl esters generated upon all-trans-retinol loading in ARPE-19 cells.

Conclusions:: ADRP in mouse RPE was localized to canonical LDs. The hydrophobic cleft of TIP47 is important for its targeting to the LDs.

Keywords: retinal pigment epithelium • protein structure/function • microscopy: light/fluorescence/immunohistochemistry