Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Imanishi, Yoshikazu; Batten, Matthew L.; Piston, David W.; Baehr, Wolfgang; Palczewski, Krzysztof

doi:10.1083/jcb.200311079

Cited by 170 publications

(232 citation statements)

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“…It has also been proposed that palmitoylation of RPE65 by LRAT regulates its subcellular localization and its ligand binding selectivity versus retinol or retinyl esters (54). We have shown here that microsome-derived RPE65 associates in a stable fashion with the ER membrane, and we have shown previously that RPE65 does not co-localize with retinyl ester-containing retinosomes (8). The RPE65 protein was not detectable in the LRAT-expressing T-REx-293 cells described here and is also absent in hepatic stellate cells.…”

Section: Discussionmentioning

confidence: 57%

“…The composition of lipid droplets of hepatic stellate cells includes many other lipid classes in addition to retinyl esters, yet the absence of LRAT affects the formation of these specialized structures (12). Retinosomes found in the RPE have been shown to be distinct from other subcellular organelles and to include proteins (such as adipose differentiation-related protein) that are involved in lipid metabolism (8). It is feasible that newly synthesized retinyl esters accumulate between the two leaflets of the ER and eventually bud off into the cytoplasm encapsulated by the cytoplasmic leaflet of the ER membrane, a possibility considered for the transport of triglycerides (9).…”

Section: Discussionmentioning

confidence: 99%

“…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 Leica TCS SP2 confocal/multiphoton microscope equipped with a titanium/sapphire laser (Chameleon TM -XR, Coherent, Inc.).…”

Section: Methodsmentioning

confidence: 99%

“…It has been shown that, in the RPE, retinyl esters are associated with retinosomes (8). Retinosomes are distinct from other subcellular organelles such as the ER, Golgi, lysosomes, and mitochondria (8). We studied the activity and localization of LRAT-expressing transfected cells incubated with either all-trans-retinol or oleic acid (Fig.…”

Section: The Putative C-terminal Transmembrane Domain Is Necessary Fomentioning

confidence: 99%

“…Recent studies have highlighted the importance of lecithin:retinol acyltransferase (LRAT) 3 in the absorption and retention of retinol in intracellular stores (7). 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). Mutations in the LRAT gene lead to early-onset severe retinal dystrophy (10).…”

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confidence: 99%

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Topology and Membrane Association of Lecithin: Retinol Acyltransferase

Moise

Golczak

Imanishi

et al. 2007

Journal of Biological Chemistry

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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.The metabolism of vitamin A (all-trans-retinol) leads to the formation of all-trans-retinoic acid and 11-cis-retinal derivatives that play crucial roles in such processes as development, immunity, and vision (1-6). Recent studies have highlighted the importance of lecithin:retinol acyltransferase (LRAT) 3 in the absorption and retention of retinol in intracellular stores (7). 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). Mutations in the LRAT gene lead to early-onset severe retinal dystrophy (10). Disruption of the Lrat gene in Lrat Ϫ/Ϫ mice produces a severe impairment in retinol uptake and storage capacity. As a result, Lrat Ϫ/Ϫ mice are blind (11) and more susceptible to vitamin A deficiency than their wild-type (WT) counterparts (12). Lrat Ϫ/Ϫ mice possess only trace amounts of retinyl esters in most tissues (11, 12), yet, surprisingly, the levels of retinyl esters in their adipose tissue are elevated compared with those in WT mice (12). Adipose stores of retinyl esters could depend on the activity of acyl-CoA:retinol acyltransferase (12).LRAT plays a pivotal role in determining retinol availability, which in turn affects the levels of all-trans-retinoic acid, a potent regulator of the expression of many genes via retinoic acid receptors (13). Expression of LRAT in the liver is influenced by multiple factors, including vitamin A status (14), retinoic acid (15), other synthetic retinoic acid receptor activators (16), and peroxisome proliferator-activated receptor ␤/␦ agonists (17). Thus, regulation of LRAT activi...

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Section: Discussionmentioning

confidence: 57%