Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy

Abstract

We have used freeze-fracture electron microscopy to examine the oligomeric structure and molecular asymmetry of integral plasma membrane proteins. Recombinant plasma membrane proteins were functionally expressed in Xenopus laevis oocytes, and the dimensions of their freeze-fracture particles were analyzed. To characterize the freeze-fracture particles, we compared the particle cross-sectional area of proteins with alpha-helical transmembrane domains (opsin, aquaporin 1, and a connexin) with their area obtained from existing maps calculated from two-dimensional crystals. We show that the cross-sectional area of the freeze-fracture particles corresponds to the area of the transmembrane domain of the protein, and that the protein cross-sectional area varies linearly with the number membrane-spanning helices. On average, each helix occupies 1.40 +/- 0.03 nm2. By using this information, we examined members from three classes of plasma membrane proteins: two ion channels, the cystic fibrosis transmembrane conductance regulator and connexin 50 hemi-channel; a water channel, the major intrinsic protein (the aquaporin 0); and a cotransporter, the Na+/glucose cotransporter. Our results suggest that the cystic fibrosis transmembrane conductance regulator is a dimer containing 25 +/- 2 transmembrane helices, connexin 50 is a hexamer containing 24 +/- 3 helices, the major intrinsic protein is a tetramer containing 24 +/- 3 helices, and the Na+/glucose cotransporter is an asymmetrical monomer containing 15 +/- 2 helices.

Figure 1

P face freeze-fracture micrographs of the plasma membrane of X. laevis oocytes. (A) The P face of control oocytes shows a low density of particles at 355 ± 21/μm² (n = 2,130). (B) In an oocyte expressing SGLT1, the density of the particles increased to 3,287 ± 112/μm² (n = 2,565). (C) High-magnification representative images of endogenous P face particles, AQP1, the short axis of opsin, the long axis of opsin, Cx50, MIP, SGLT1, and CFTR. [Bars = 100 nm (A and B) and 20 nm (C).]

Figure 2

Size distribution of P face endogenous particles. With respect to size, control oocytes exhibit a fairly uniform particle population; ≈93% have a mean diameter of 7.6 ± 0.5 nm, and ≈7% measure 11.1 ± 1.0 nm (N = 875).

Figure 3

Size distribution of P face particles in oocytes expressing AQP1, opsin, and Cx50. (A) In an oocyte expressing AQP1, there was a population of particles with a mean diameter of 8.9 ± 0.2 nm (N = 449) in addition to the endogenous particles. The particle density in this oocyte was 825 ± 63/μm² (n = 982). In oocytes expressing opsin, two particle populations occurred with similar frequency: 5.7 ± 0.3 nm (43%) and 6.8 ± 0.1 nm (57%) (N = 912). Particle density was 1,444 ± 43/μm² in this oocyte (n = 1,903). Cx50 particles had a mean diameter of 9.0 ± 0.3 nm (N = 411). The particle density in this oocyte was 1,415 ± 81/μm² (n = 1,763). The small population of apparently larger size (≈11 nm) in all of the above histograms corresponds to the population of larger endogenous particles (see Fig. 2). Cross-hatched regions correspond only to the 7.6-nm P face endogenous particles as confirmed by particle density determinations. (B) Relationship between the transmembrane domain cross-sectional area and the number of transmembrane α-helices. Freeze-fracture data for AQP1, opsin, and Cx50 (•) agree well with those from two-dimensional crystals of AQP1, opsin, and Cx43 (○) (1.40 ± 0.03 vs. 1.42 ± 0.05 nm²/helix).

Figure 4

Size distribution of P face particles in oocytes expressing MIP, SGLT1, and CFTR. (A) MIP particles demonstrated a single population at 9.0 ± 0.3 nm (N = 430). The particle density in this oocyte was 3,288 ± 136/μm² (n = 2,878). SGLT1 particles appeared at 7.1 ± 0.2 (48%) and 8.2 ± 0.4 nm (52%) (N = 640). In this oocyte, the particle density was 2,070 ± 130/μm² (n = 2,163). CFTR particles were 9.0 ± 0.2 nm in diameter (N = 500). A larger particle (11.0 ± 0.8 nm) appeared in the CFTR frequency histogram that coincides with the larger endogenous particles but was present at a higher relative frequency (≈10% of exogenous particles). Particle density was 678 ± 27/μm² (n = 944). Cross-hatched regions correspond only to the 7.6-nm P face endogenous particles as confirmed by particle density determinations. (B) Relationship between the transmembrane domain cross-sectional area and the number of transmembrane α-helices. The straight line corresponds to the linear regression through the AQP1, opsin, and Cx50 data (•) (slope, 1.40 ± 0.03 nm²/helix). ○, Calculated cross-sectional areas of MIP, SGLT1, and CFTR.