Transmembrane organization of the Na,K-ATPase determined by epitope addition
1Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Abstract:
The Na,K-ATPase is a membrane-associated enzyme that establishes the internal Na+/K+ environment of most animal cells. The catalytic (alpha) subunit of the Na,K-ATPase contains multiple transmembrane segments, but the number and location of these domains has not been clearly established. We have used epitope addition to determine the transmembrane topology of the alpha subunit. An immunoreactive peptide was inserted into various regions of the cDNA encoding the rat alpha 1 subunit, and the constructs were expressed in transfected mammalian cells. The intra- or extracellular location of the epitope tags was determined by immunofluorescence analysis. Our results indicate that the amino and carboxyl termini of the alpha subunit are situated intracellularly, and the polypeptide is likely to possess eight membrane-spanning segments. The systematic application of epitope tagging may be useful for analyzing the topology of membrane proteins of unknown structure.
Related Concept Videos
Translocation of Proteins into the Mitochondria
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Protein Transport into the Inner Mitochondrial Membrane
Transport of mitochondrial precursors across the TIM23 channel is driven by...
Insertion of Single-pass Transmembrane Proteins in the RER
Integral transmembrane proteins possess transmembrane and extra membrane domains. The transmembrane domains are primarily made of 20-25 hydrophobic amino acids arranged in a helical secondary confirmation. These...
Insertion of Multi-pass Transmembrane Proteins in the RER
The multipass transmembrane proteins are the type IV integral membrane proteins with multiple topogenic sequences determining their spatial arrangement in the ER membrane. Nearly all multipass proteins lack a cleavable signal sequence and use...
Tail-anchoring of Proteins in the ER Membrane
Membrane Asymmetry Regulating Transporters
Flippase
Eukaryotic flippases are type-IV P-type ATPases or P4-ATPases belonging to P-type ATPase family proteins that are membrane-bound pumps involved in the ATP-mediated transport of ions and molecules across the membrane. Flippases flip specific phospholipids from the outer to the inner leaflet of a membrane. All P4-ATPases have one...


