SURFACE TOPOGRAPHIES AT SUBNANOMETER-RESOLUTION REVEAL ASYMMETRY AND SIDEDNESS OF AQUAPORIN-1

Aquaporin-1 (AQP1) is an abundant protein in human erythrocyte membran es which functions as a specific and constitutively active water condu cting pore. Solubilized and isolated as tetramer, it forms well-ordere d two-dimensional (2D) crystals when reconstituted in the presence of lipids. Several high resolution projection maps of AQP1 have been dete rmined, but information on its three-dimensional (3D) mass distributio n is sparse. Here, we present surface reliefs at 0.9 nm resolution tha t were calculated from freeze-dried unidirectionally metal-shadowed AQ P1 crystals as well as surface topographs recorded with the atomic for ce microscope of native crystals in buffer solution. Our results confi rm the 3D map of negatively stained AQP1 crystals, which exhibited tet ramers with four major protrusions on one side and a large central cav ity on the other side of the membrane. Digestion of AQP1 crystals with carboxypeptidase Y, which cleaves off a 5 kDa intracellular C-termina l fragment, led to a reduction of the major protrusions, suggesting th at the central cavity of the tetramer faces the outside of the cell. T o interpret the results, sequence based structure predictions served a s a guide. (C) 1996 Academic Press Limited.