Topology and membrane association of lecithin: retinol acyltransferase.
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.
Moise
Alexander R
AR
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
Golczak
Marcin
M
Imanishi
Yoshikazu
Y
Palczewski
Krzysztof
K
D000217:Acyltransferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D014415:Tunicamycin / Q000494:pharmacology; D014801:Vitamin A / Q000737:chemistry
D000900:Anti-Bacterial Agents; D000911:Antibodies, Monoclonal; D010713:Phosphatidylcholines; D014415:Tunicamycin; D014801:Vitamin A; D000217:Acyltransferases; C056942:lecithin-retinol acyltransferase