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Fig. 1
STRA6-Dependent Uptake and Release of Retinoids
(A) Subcellular localization of STRA6 tagged with 1D4 epitope (blue) stably expressed in NIH 3T3 cells was determined by immunohistochemistry and confocal microscopy; calreticulin was detected by anti-calreticulin polyclonal antibody (red). Scale bar = 20 μm.
(B) Chromatograms representing retinoid composition of STRA6-, LRAT-, and STRA6/LRAT-expressing cells after incubation with 8 μM holo-RBP4. Asterisk indicates the position at which all-trans-retinol migrates.
(C) Time course of retinol uptake. LRAT- and STRA6/LRAT-expressing cells were incubated with 8 μM holo-RBP4. Retinoid content of the cells was determined at the time points indicated. Values represent the mean ± SD of three independent experiments.
(D) STRA6-dependent release of retinol. Cells were incubated with 10 μM all-trans-retinol for 16 hr prior to this experiment. Cells then were washed with PBS, and fresh medium containing 8 μM apo-RBP4 was added. After 16 hr, the medium was collected and retinoid composition was determined by HPLC. Values represent the mean ± SD of three independent experiments.
(E) Retinoic acid uptake is not STRA6 dependent. Apo-RBP was loaded with all-trans-retinoic acid (RA) and purified by fast protein liquid chromatography (FPLC). Efficiency of RA loading was confirmed by absorption spectroscopy (inset). NIH 3T3 cells stably expressing STRA6 and LRAT were incubated with 8 μM RA-RBP4 or 8 μM RA overnight. Retinoid composition was determined by reverse-phase chromatography.
Reprinted from Cell Metabolism, 7(3), Isken, A., Golczak, M., Oberhauser, V., Hunzelmann, S., Driever, W., Imanishi, Y., Palczewski, K., and von Lintig, J., RBP4 Disrupts Vitamin A Uptake Homeostasis in a STRA6-Deficient Animal Model for Matthew-Wood Syndrome, 258-268, Copyright (2008) with permission from Elsevier. Full text @ Cell Metab.