Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Yoshikazu Imanishi,Marcin Golczak,Krzysztof Palczewski,Alexander R Moise

doi:10.1074/jbc.m608315200

Yoshikazu Imanishi, Marcin Golczak + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m608315200

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Abstract

Fatty acid retinyl esters are the storage form of vitamin A (all-trans-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.

Highlights

Lecithin:retinol acyltransferase (LRAT) catalyzes the synthesis of retinyl esters, thereby drawing retinol from the circulation to storage depots such as the lipid droplets of hepatic stellate cells and the retinosome structures found in the retinal pigment epithelium (RPE) [8, 9]
To detect anti-LRAT immunoreactivity, we rinsed sections in PBST three times and incubated them with either Cy3conjugated 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
Our analysis led to the hypothesis that LRAT is a single membrane-spanning protein anchored to the endoplasmic reticulum (ER) membrane via the C-terminal transmembrane domain and assuming an N-terminal cytoplasmic/C-terminal luminal orientation

Summary

Introduction

LRAT Is an Endoplasmic Reticulum (ER)-localized Membrane Protein—Hydropathy analysis of the sequence of mouse LRAT indicated that there is only one region at the C terminus of the protein that has high probability of being a transmembrane domain (Fig. 1A). Mouse tLRAT protein expressed in bacterial cells exhibited robust acyltransferase activity, indicating that its catalytic domain is located in the region encompassing residues 35–195 (data not shown).

Results

Conclusion