Abstract
Abstract: :
Purpose: Recent studies have revealed that RPE65, a major peripheral membrane protein of the retinal pigment epithelium, stereospecifically binds all–trans–retinyl esters and allows them to enter the visual cycle (1–3). However, what governs membrane association of RPE65, and the nature of possible post–translational modifications relevant to this remain obscure. Accurate molecular weight measurements of RPE65 revealed two classes of RPE65, one of which was membrane associated (4). Here, we report on the post–translational modifications of RPE65 which allow it to associate with membranes. Methods: In this study, bovine RPE65 is purified in both membrane–bound (mRPE65) and soluble forms (sRPE65). 2D SDS–PAGE further separates mRPE65 based on pI differences. Mass spectrometric analysis (ESI–MS/MS and MALDI–TOF), coupled with biochemical studies (reducing vs. non–reducing condition and cysteine blocking,) reveals several palmitoylated species. Results: A total of five palmitoyl residues are adducted to either serine, threonine, or cysteine residues in mRPE65, whereas only one residue is palmitoylated in sRPE65. N–terminal acetylation and one disulfide bond are also detected in RPE65. Conclusions: These studies demonstrate that RPE65 is post–translationally modified by N–terminal acetylation and poly–palmitoylation. The covalently attached palmitoyl groups at the Cys/Ser/Thr residues determine the state of membrane association of RPE65. References: 1. Jahng, W. J. David, C., Nesnas, N., Nakanishi, K., and Rando, R. R. (2003) Biochemistry 42, 6159–6168. 2. Gollapalli D. R., Maiti, P., and Rando, R. R. (2003) Biochemistry 42 11824–11830. 3. Mata, N. L., Moghrabi, W. N., Lee, J. S., Bui, T. V., Radu, R. A., Horwitz, J., Travis, G. H. (2003) J. Biol. Chem. in press. 4. Ma, J.–x., Zhang, J., Othersen, K. L., Moiseyev, G., Ablonczy, Z., Redmond, T. M., Chen, Y., Crouch, R. K. (2001) Invest. Ophthalmol. Vis. Sci. 42, 1429–1435.
Keywords: retinal pigment epithelium • protein modifications–post translational • protein purification and characterization