Figure 1.
Effects of acute chemical modification on STRA6-catalyzed retinol release from holo-RBP.
STRA6-catalzyed retinol release from holo-RBP causes a decline in retinol fluorescence. In all experiments, holo-RBP was added at 0 min. A and B. Effects of MTSEA-biotin treatment on the retinol release activity of STRA6-WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in A (open circles). C. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C. Activity of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. D. Structures of MTSEH, MTSBS, MTSPT and MTSEA-biotin. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of S385C. The color of the trace in E matches the color of the chemical in D. Grey trace, no treatment. F. Quantitation of retinol release activity of experiments in E. Activity of the untreated reaction is defined as 1.
Figure 2.
Real-time analysis of STRA6-catalyzed transport of retinol from holo-RBP to EGFP-CRBP-I.
A. Schematic diagram of the use of retinol-EGFP FRET to monitor the transport of retinol from holo-RBP to EGFP-CRBP-I. FRET is indicated by an orange arrow. B and C. Effects of MTSEA-biotin treatment on the retinol transport by STRA6 WT and STRA6-S385C, respectively. Grey traces, no treatment. Green traces, MTSEA-biotin treatment. Control membrane without STRA6 was used as the negative control in B (open circles). D. Comparison of the effects of MTSEA-biotin on STRA6-WT and STRA6-S385C at 60 min. The signal of the untreated reaction for STRA6-WT is defined as 1. Grey bars, no treatment. Green bars, MTSEA-biotin treatment. E. Comparison of effects of MTSEH, MTSBS, MTSPT and MTSEA-biotin of retinol transport activity of STRA6-S385C. F. Quantitation of the FRET signals from experiments in E. The signal of the untreated reaction is defined as 1.
Figure 3.
Effect of MTSEA-biotin modification on STRA6-mediated vitamin A uptake as measured by 3H-retinol uptake from 3H-retinol/RBP and HPLC analysis of retinyl esters.
A. Positions of V320C and S385C in the transmembrane topology model of STRA6. Because MTSEA-biotin is membrane impermeable, the location of S385C close to the intracellular side of STRA6 makes it inaccessible to MTSEA-biotin from the extracellular side, while V320C is positioned on the boundary of membrane helix VI and extracellular domain and can be accessed by MTSEA-biotin from the extracellular side. B and C. Cell-free vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-S385C (S385C). B is 3H-retinol uptake assay from 3H-retinol/RBP. C is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. D and E. Live-cell vitamin A uptake assays comparing the effect of MTSEA-biotin treatment on STRA6-WT (WT) and STRA6-V320C (V320C). D is 3H-retinol uptake assay from 3H-retinol/RBP. E is HPLC-based retinyl ester analysis of retinol uptake from holo-RBP. In B and D, the amount of 3H-retinol associated with STRA6-WT without modification is defined as 100%. This activity reflects the binding of 3H-retinol/RBP to STRA6. In C and E, the activity of STRA6-WT with LRAT as measured by retinyl ester amount is defined as 100%. Grey bars, no treatment. Green bars, MTSEA-biotin treatment.
Figure 4.
Scanning the sixth transmembrane helix of STRA6 for positions that enhance the sensitivity of STRA6 to MTSEA-biotin modification.
Thirty two residues in or near the sixth transmembrane domain of STRA6 were each changed to cysteine. Each mutant was tested for STRA6-catalyzed retinol release from holo-RBP. Grey traces, no modification. Red traces, MTSEA-biotin modification. All experiments were done in triplicate.
Figure 5.
Scanning the seventh transmembrane helix of STRA6 for positions that enhance the sensitivity of STRA6 to MTSEA-biotin modification.
Twenty eight residues in or near the seventh transmembrane domain of STRA6 were each changed to cysteine. Each mutant was tested for STRA6-catalyzed retinol release from holo-RBP. Grey traces, no modification. Red traces, MTSEA-biotin modification. All experiments were done in triplicate.
Figure 6.
Comparison and quantitation of the acute suppression by MTSEA-biotin for all STRA6 cysteine mutants produced and analyzed in the study.
Suppression of the wild-type control is defined as 1. A. Mutations in or near the sixth transmembrane helix. B. Mutations in or near the seventh transmembrane helix. Statistical analysis is shown as *** (p<0.001), ** (p<0.01), or * (p<0.05). Mutants not labeled were not statistically different from the wild-type control.
Figure 7.
Schematic diagrams of the locations of key positions in STRA6 whose acute modifications block vitamin A transport by STRA6.
A. Helical wheel presentations of residues L293 to L303 and residues F379 to H389. Residues whose modification by MTSEA-biotin impedes vitamin A transport by STRA6 are labeled in light red. These key residues are located on the same side of the helix. B. Locations of the key residues in the transmembrane topology model of STRA6. Residues whose acute modification impedes vitamin A transport by STRA6 are represented as red circles.