Membrane protein isolation and structure determination in cell-derived membrane vesicles

Abstract

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca²⁺ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.

Keywords: cell membrane vesicle; cryo-EM; ion channel; membrane protein; proteoliposome.

Fig. 1.

Preparation of Slo1-containing vesicles from the total cell membrane. (A) Construct design of the human Slo1 channel. The extracellular ALFA tag is colored in magenta, and the intracellular GFP tag is colored in green. (B) Purification procedure of Slo1-containing vesicles from the total membrane. The endoplasmic reticulum (ER) membrane is colored in light gray, and the plasma membrane (PM) is colored in dark gray. (C) SDS-PAGE of the total membrane vesicles enriched with Slo1. (D) Representative micrograph of Slo1-containing vesicles from the total membrane. (E) 2D class averages of Slo1 channel from the total cell membrane.

Fig. 2.

Structural analysis of Slo1 from the total membrane vesicles. (A) Overall cryo-EM map of Slo1 from the total membrane vesicles. The protomers in Slo1 are colored in dark and light blue. The lipid bilayer (yellow) is contoured at a low threshold to show the curvature of the membrane. (B) Two-fold symmetry of Slo1 from the total membrane vesicles. Slabs of cryo-EM density parallel to the membrane plane are taken at the intracellular-most end of the Slo1 gating ring (slab 1) and the pore region (slab 2). (C) Structural comparison of the two protomers from neighboring subunits. The structures are superposed according to the selectivity filter and the pore helix. (D) Two-fold symmetric structure of Slo1 determined in detergent using the same EDTA-free buffer. The slab of cryo-EM density at the same region in B is shown. The density at the center of the pore entryway in slab 2 is likely due to detergent molecules.

Fig. 3.

Preparation of Slo1-containing vesicles from the plasma membrane. (A) Purification procedure of Slo1-containing vesicles from the plasma membrane (PM). The endoplasmic reticulum (ER) membrane is colored in light gray, and the plasma membrane is colored in dark gray. (B) SDS-PAGE of the PM vesicles enriched with Slo1. (C) Western blot showing the relative abundance of the PM marker (Na⁺/K⁺ ATPase) and the ER marker (ERp57). Normalized PM marker levels for the two different samples are shown as bar graph, beneath (n = 3). (D) Representative micrograph of Slo1-containing vesicles from the plasma membrane. (E) 2D class averages of the Slo1 channel from the plasma membrane vesicles.

Fig. 4.

Structural analysis of Slo1 from the plasma membrane vesicles. (A) Overall cryo-EM map of Slo1 from the total membrane vesicles. The protomers in Slo1 are colored in dark and light blue. The lipid bilayer (yellow) is contoured at a low threshold to show the curvature of the membrane. (B) Four-fold symmetry of Slo1 from the plasma membrane vesicles. Slabs of cryo-EM density parallel to the membrane plane are taken at the intracellular-most end of the Slo1 gating ring indicated in A. (C) The two Ca²⁺-binding sites and the Mg²⁺-binding sites are occupied. The Ca²⁺ ion is colored in orange, and the Mg²⁺ ion is colored in purple.

Fig. 5.

New structural features of Slo1 observed in plasma membrane vesicles. (A) Global conformational change of the Slo1 transmembrane domain. The models are superposed according to the transmembrane domain. The Slo1 model determined in plasma membrane (PM) vesicles is colored in blue, and the Slo1 model determined in detergent is colored in gray. A magnified view of the S0 helix and its cryo-EM density are shown on the Right. (B) Lipid (phospholipid and cholesterol) molecules observed in the cryo-EM map. The Slo1 model is colored in two shades of blue, and the cryo-EM densities for the lipid molecules are colored in light yellow. (C) Cholesterol molecules identified in the cryo-EM map. Cryo-EM densities for putative cholesterol molecules are colored in light yellow. (D) Phospholipid-binding site at the S4-S5 linker. The cryo-EM density for the phospholipid is colored in light yellow. (E) New cation (likely Na⁺) binding site identified in the Slo1 structure from plasma membrane vesicles. The Na⁺ ion is colored in light blue, and the Ca²⁺ ion is colored in orange. Right side is a close-up view of the magenta box on the left.The atomic model and cryo-EM density map are shown as wall-eyed stereoscopic images.