Topology of the Na,K-ATPase. Evidence for externalization of a labile transmembrane structure during heating.
The topological organization of the Na,K-ATPase alpha subunit is controversial. Detection of extracellular proteolytic cleavage sites would help define the topology, and so attempts were made to find conditions and proteases that would permit digestion of Na,K-ATPase in sealed right-side-out vesicles from renal medulla. The beta subunit is predominantly extracellular and could mask the surface of the alpha subunit. Most of the tested proteases cleaved beta, and some digested it extensively. However, without further disruption of structure, there was still no digestion of the alpha subunit. Reduction (at 50 degrees C) of disulfide bonds that might stabilize the beta subunit fragments, or heating alone at 55 degrees C, permitted tryptic digestion of alpha at a site close to the C terminus, while simultaneously increasing digestion of beta. A 90-kDa N-terminal fragment of alpha was recovered, but the C-terminal fragment was further digested. Heating and reduction resulted in the extracellular exposure of a protein kinase A phosphorylation site, Ser-938, and the C terminus, both of which have been proposed to be located on the intracellular surface. At the same time, access to a distant protein kinase C phosphorylation site was not increased. The data suggest that the harsh treatment simultaneously resulted in alteration of the beta subunit and the extrusion of a segment of alpha that normally spans the membrane, without causing complete denaturation or opening the sealed vesicles. Preincubation with Rb+ was protective, consistent with prior evidence that it stabilizes the protein segments in the C-terminal third of alpha. We conclude that this portion of the alpha subunit contains a transmembrane structure with unique lability to heating.
Arystarkhova
E
E
Laboratory of Membrane Biology, Massachusetts General Hospital, Charlestown 02129, USA.
Gibbons
D L
DL
Sweadner
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008024:Ligands; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011487:Protein Conformation; D051381:Rats; D012413:Rubidium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D008024:Ligands; D017868:Cyclic AMP-Dependent Protein Kinases; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium