The assembly of 16-kDa polypeptide channel units in membranes from the hepatopancreas of Nephrops norvegicus has been studied both by electron microscopy and by the lipid-protein interactions reported with spin-labeled lipids. Membranes prepared by extraction with N-lauroylsarcosine and Triton X-100 have a low lipid/protein ratio (ca. 4-6.5 phospholipids and 1 cholesterol per 16-kDa monomer), and those prepared by alkaline extraction have a higher lipid/protein ratio (ca. 12-16 phospholipids and 3.5-4 cholesterols per 16-kDa monomer). In the membranes extracted with detergents, the protein is assembled in membrane sheets as hexagonally packed hexameric complexes, whereas the alkali-extracted preparations consist of closed vesicles in which the channel complexes are near randomly distributed. The electron spin resonance (ESR) spectra from lipids spin-labeled at the C-14 position of the (sn-2) chain show lower mobility for the membranes extracted with A-lauroylsarcosine than for the alkalineextracted membranes. At higher temperatures, the ESR spectra reveal a population of lipids whose mobility is restricted by direct interaction with the intramembranous sections of the channel assemblies. The population of protein-associated spin-labeled phosphatidylcholine in the alkali-extracted membranes corresponds to 4-5 phospholipid molecules plus 1 cholesterol molecule per 16-kDa polypeptide monomer. These numbers are only slightly smaller than the number of lipid molecules that can be accommodated around the perimeter of the model proposed for the channel complex [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] that consists of a hexameric arrangement of transmembrane four-helix bundles. The 16- kDa polypeptide displays a selectivity relative to phosphatidylcholine for the negatively charged spinlabeled lipids, stearic acid, phosphatidylserine, and phosphatidylglycerol, suggesting that basic amino acid residues (possibly Lys-53, Lys-78, Lys-156, Arg-120, and Arg-127) are located close to the lipid headgroups in the channel assembly.
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