Evidence for a common structure for a class of membrane channels

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Abstract
Electron microscopic analysis of gap-junction-like structures isolated from an arthropod (Nephrops norvegicus) and composed of a 16-kDa polypeptide, show the functional unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16–18 kDa. Fourier transform infrared spectroscopy indicates a high α-helical content for the protein, supporting secondary-structure predictions of four transmembrane α helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four α-helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7–15]. The reconstructed image is also similar to that obtained for gap-junction-like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255–263] whose protein content was unknown but which we demonstrate may be composed of a related 16-kDa protein. Previous studies have shown a high sequence identity of the Nephrops 16-kDa protein with the 16-kDa proteolipid subunit c of the vacuolar H+-ATPase, both of which in turn bear similarity to the 8-kDa proteolipid subunit of the F1F0-ATP synthase. Expression of cDNA coding for the Nephrops 16-kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V-ATPase proteolipid has been inactivated, restores V-ATPase activity and cell growth.