Biogenesis and topology of the secretory Na+-K+-2Cl- cotransporter (NKCC1) studied in intact mammalian cells.
The "secretory" Na+-K+-2Cl- cotransporter (NKCC1) is a member of a small gene family with nine homologues in vertebrates. Of these, seven are known to be electroneutral chloride transporters. These transporters play a number of important physiological roles related to salt and water homeostasis and the control of intracellular chloride levels. Hydropathy analyses suggest that all of these transporters have a similar transmembrane topology consisting of relatively large intracellular N and C termini and a central hydrophobic domain containing 12 membrane-spanning segments (MSSs). In recent experiments from our laboratory [Gerelsaikhan, T., and Turner, R. J. (2000) J. Biol. Chem. 275, 40471-40477], we employed an in vitro translation system to confirm that each of the putative MSSs of NKCC1 was capable of membrane integration in a manner consistent with a 12 MSS model. Here, we extend that work to the study of the biogenesis of NKCC1 in intact cells. We employ a truncation mutant approach that allows us to monitor this process quantitatively as successive MSSs are synthesized. While the results presented here confirm the 12 MSS model, they also indicate that the integration of NKCC1 into the membrane does not occur via a simple cotranslational process. In particular, we demonstrate that two MSSs, the second and sixth, require the presence of downstream sequence to efficiently integrate into the membrane.
Gerelsaikhan
Tudevdagva
T
Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, DHHS, Bethesda, Maryland 20892, USA.
Parvin
Most Nahid
MN
Turner
R James
RJ
D000595:Amino Acid Sequence / Q000235:genetics*; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D017384:Sequence Deletion; D028021:Sodium-Potassium-Chloride Symporters / Q000096:biosynthesis* / Q000235:genetics; D064506:Solute Carrier Family 12, Member 2
C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2