Purpose

To determine molecular mechanisms for the release of 11-cis-retinal from the binding pocket of cellular retinaldehyde-binding protein (CRALBP).

Methods

Binding of CRALBP to lipid surfaces was assessed with a lipid-immunoblot assay. Lipids were presented to CRALBP as small unilamellar vesicles (SUVs) consisting of phosphatidylcholine (PC) plus other lipids. Release of 9-cis-retinal or 11-cis-retinal from CRALBP was measured with spectral and high performance liquid chromatography (HPLC) assays based on the protection of the protein-bound retinal carbonyl group from reaction with NH 2 OH. The electrostatic surface potential of CRALBP was calculated from a model of its structure using the program CCP4mg.

Results

Incubation of CRALBP· 11-cis-retinal with lipids absorbed on nitrocellulose revealed binding to the acidic lipids, phosphatidic acid (PA)> phosphatidylinositol 3, 4, 5-trisphosphate [PI (3, 4, 5) P 3]> phosphatidylserine (PS)> PI (4, 5) P 2 and little or no binding to PC, phosphatidylethanolamine (PE), or PI (4) P. 11-cis-retinal was released during incubation of CRALBP with SUVs consisting of PC plus 50 mol% PA but not during incubation with those composed of 100 mol% PC. The efficacy of release of 9-cis-retinal or 11-cis-retinal from CRALBP by phospholipid-containing SUVs generally paralleled that of the binding of CRALBP to the lipids (PA> PS> PI>> PC). Examination of the electrostatic surface potential of the protein structure revealed a basic recess on one face of the protein, which may bind acidic lipids.

Conclusions

Our results identify the first physiologic substances that release 11-cis-retinal from CRALBP. PA and PS are relatively minor membrane lipids that can be generated in the cytoplasmic leaflet of the plasma membrane in response to various signal transduction pathways, where they could interact with cytosolic CRALBP. The mechanism for release of retinal from CRALBP by acidic lipids remains to be determined but could involve binding of the acidic lipid in the 11-cis-retinal binding site or to the positive basic recess on the protein surface. These results open a new facet in our understanding of how CRALBP functions in the regeneration of visual pigments.