Topological analysis of NHE1, the ubiquitous Na+/H+ exchanger using chymotryptic cleavage.
Proteases, glycosidases, and impermeant biotin derivatives were used in combination with antibodies to analyze the subcellular distribution and transmembrane disposition of the Na+/H+ exchanger NHE1. Both native human NHE1 in platelets and epitope-tagged rat NHE1 transfected into antiport-deficient cells were used for these studies. The results indicated that 1) the entire population of exchangers is present on the surface membrane of unstimulated platelets, ruling out regulation by recruitment of internal stores of NHE1; 2) the putative extracellular loops near the NH2 terminus are exposed to the medium and contain all the N- and O-linked carbohydrates; 3) by contrast, the putative extracellular loops between transmembrane domains 9-10 and 11-12 are not readily accessible from the outside and may be folded within the protein, perhaps contributing to an aqueous ion transport pathway; 4) the extreme COOH terminus of the protein was found to be inaccessible to extracellular proteases, antibodies, and other impermeant reagents, consistent with a cytosolic localization; and 5) detachment of approximately 150 amino acids from the NH2-terminal end of the protein had little effect on the transport activity of NHE1.
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
L D
LD
Shrode
B S
BS
Gan
S J
SJ
D'Souza
J
J
Orlowski
S
S
Grinstein
D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry; D002918:Chymotrypsin; D003600:Cytosol / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010449:Peptide Mapping; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D013450:Sulfones / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D013450:Sulfones; C069139:growth factor-activatable Na-H exchanger NHE-1; C074107:3-methylsulfonyl-4-piperidinobenzoyl guanidine; D000584:Amiloride; D002918:Chymotrypsin