Technical Reference #3119

3119.

Topology and Membrane Association of Lecithin: Retinol Acyltransferase Alexander R. Moise;Marcin Golczak; Yoshikazu Imanishi; and Krzysztof Palczewski, Case Western Reserve University, Journal of Biological Chemistry, 282(3119), (2007)
Link To Paper

Abstract:
Fatty acid retinyl esters are the storage form of vitamin A (alltrans- retinol) and serve as metabolic intermediates in the formation of the visual chromophore 11-cis-retinal. Lecithin:retinol acyltransferase (LRAT) the main enzyme responsible for retinyl ester formation acts by transferring an acyl group from the sn-1 position of phosphatidylcholine to retinol. To define the membrane association and localization of LRAT we produced an LRAT-specific monoclonal antibody which we used to study enzyme partition under different experimental conditions. Furthermore we examined the membrane topology of LRAT through an N-linked glycosylation scanning approach and protease protection assays. We show that LRAT is localized to the membrane of the endoplasmic reticulum (ER) and assumes a single membrane-spanning topology with an N-terminal cytoplasmic/C-terminal luminal orientation. In eukaryotic cells the C-terminal transmembrane domain is essential for the activity and ER membrane targeting of LRAT. In contrast the N-terminal hydrophobic region is not required for ER membrane targeting or enzymatic activity and its amino acid sequence is not conserved in other species examined. We present experimental evidence of the topology and subcellular localization of LRAT a critical enzyme in vitamin A metabolism.

Materials & Methods:
Two-photon Vitamin A Imaging and Immunohistochemistry— RPEcells obtained fromWTor Lrat / mice were stained using the anti-LRAT monoclonal antibody (11). We transiently transfected COS-7 cells to study the targeting and distribution of WT LRAT and its truncation mutants. Formation of retinosomes (retinyl ester storage particles) was studied in vitro in stably transfected HEKK-LRAT cells which express mouse WT LRAT under the control of a tetracycline-inducible promoter (32). Cells were cultured in complete Dulbecco’s modified Eagle’s medium on glass-bottom microwell dishes (MatTek Corp. Ashland MA). Expression of LRAT was induced in HEKK-LRAT cells with tetracycline added 48 h before analysis. The medium was supplemented 24 h before analysis with alltrans- retinol (10 M) or oleic acid (0.1 mM). Medium containing substrates was overlaid on cells and incubated overnight in the dark at 37 °C in 5% CO2 under 100% humidity. Immunohistochemical staining was performed by fixing HEKK-LRAT cells or transiently transfected COS-7 cells with 4% paraformaldehyde in phosphate-buffered saline (PBS; 136mM NaCl and 11.4 mM sodium phosphate (pH 7.4)) for 10 min. Cells were washed three times with PBST (PBS with 0.1% Triton X-100) followed by incubation in 1.5% normal goat serum in PBST for 15 min at room temperature to block nonspecific binding. Staining was done overnight at 4 °C with the anti-LRAT monoclonal antibody (11) a goat anti-BiP polyclonal antibody (Santa Cruz Biotechnology Inc.) and/or the rabbit anti-calreticulin polyclonal antibody. Cells were rinsed in PBST three times and incubated with Alexa 488-conjugated donkey anti-goat IgG (Invitrogen) for detection of anti-BiP immunofluorescence or with Cy3- conjugated goat anti-rabbit IgG for detection of calreticulin. To detect anti-LRAT immunoreactivity we rinsed sections in PBST three times and then incubated them with either Cy3- conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.) for the results presented in Fig. 3 or with Alexa 488-conjugated goat anti-mouse IgG for the results presented in Figs. 2 5 and 7. Cells were mounted in 2% 14- diazabicyclo[222]octane in 90% glycerol to retard photobleaching. For detection of neutral lipid we incubated cells with 10 g/ml Nile red (9-diethylamino-5H-benzo[ ]phenoxazine-5- one; Invitrogen) in PBS. Intrinsic fluorescence of retinyl esters was visualized by two-photon microscopy (8). The images were obtained using either a confocal/two-photon microscope (Zeiss LSM 510 MP-NLO) with a mode-locked titanium/sapphire laser (Mira 900 Coherent Inc. Mountain View CA) or a LeicaTCSSP2 confocal/multiphoton microscope equipped with a titanium/sapphire laser (ChameleonTM-XR Coherent Inc.).

Microscopic Technique
two-photon microscopy

Cell Type(s)
RPEcells; COS-7 cells; HEKK-LRAT cells