Abstract
STRA6 (stimulated by retinoic acid 6) is a 75kDa polytopic transmembrane protein that facilitates cellular retinol uptake from retinol-binding protein (RBP). Structural characterization of STRA6 from Danio rerio purified in detergent and reconstituted in amphipol A8-35 was achieved by single-particle cryo-electron microscopy (cryo-EM). This provided the first high-resolution snapshot of this protein, showing a novel topology of a tightly assembled homodimer, and an unexpected physiological association with calmodulin in addition to insights into its potential mechanism of function. Specifically, a large hydrophobic cavity in the center of STRA6 linked to the known site of interaction with RBP suggested a route of retinol entry into the cell by diffusion into the membrane through a lateral opening of the cavity directly into the bilayer. Moreover, the capability to produce pure and homogeneous protein has allowed previously unattainable functional characterization of STRA6 in a reconstituted system. Here, we describe detailed methods for Danio rerio STRA6 expression in insect cells, purification in detergent and reconstitution in amphipol for structural characterization by cryo-EM. Furthermore, we show reconstitution of the protein in liposomes for an in vitro proteoliposome-based assay of STRA6-mediated retinol uptake. Finally, we present methods and preliminary cryo-EM data for STRA6 incorporated in lipid-filled nanodiscs, a close to native milieu to study membrane protein structure and function.