Transmembrane topology of the secretory Na+-K+-2Cl- cotransporter NKCC1 studied by in vitro translation.
The secretory Na(+)-K(+)-2Cl(-) cotransporter NKCC1 is a member of a small gene family of electroneutral salt transporters. Hydropathy analyses indicate that all of these transporters have a similar general structure consisting of large hydrophilic N and C termini on either side of a central, relatively well conserved, hydrophobic domain. Programs that predict the transmembrane topology of polytopic membrane proteins identify 10-12 putative membrane-spanning segments (MSSs) in this hydrophobic domain; but to date, there is little experimental data on the structure of this region for any of these transporters. In this report, we have studied the transmembrane topology of NKCC1 using an in vitro translation system designed to test the membrane insertion properties of putative MSSs (Bamberg, K., and Sachs, G. (1994) J. Biol. Chem. 269, 16909-16919). Fusion proteins consisting of putative NKCC1 MSSs inserted either (i) between an N-terminal cytosolic anchor sequence and a C-terminal reporter sequence containing multiple N-linked glycosidation sites or (ii) between an N-terminal signal anchor sequence and the same glycosidation flag were expressed in the presence of canine pancreatic microsomes. The glycosidation status of the reporter sequence, which indicated its luminal or extraluminal location in the microsomes, was then used to characterize the signal anchor or stop transfer activity of the inserted MSSs. The results of this experimental analysis yielded a topology scheme consisting of 12 membrane-spanning segments, two pairs of which apparently form rather tight hairpin-like structures within the membrane.
Gerelsaikhan
T
T
Membrane Biology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Turner
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011188:Potassium / Q000378:metabolism; D014176:Protein Biosynthesis; D051381:Rats; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D002712:Chlorides; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D012964:Sodium; D011188:Potassium