Fast degradation of the auxiliary subunit of Na+/K+-ATPase in the plasma membrane of HeLa cells

Abstract

The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (α1) and auxiliary (β1 and β3) subunits of Na+/K+-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the β-subunit is required for the cell-surface expression of the α-subunit, the plasma membrane contains more α-subunits than β-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the β-subunits was much faster than that of the α1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the β1-subunit. Double knockdown of the β1- and β3-subunits by RNAi resulted in the disappearance of these β-subunits but not the α1-subunit in the plasma membrane. All these results indicate that the α- and β-subunits of Na+/K+-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.

A Direct Role for ATP1A1 in Unconventional Secretion of Fibroblast Growth Factor 2

“…Inhibition of FGF2 secretion in the absence of ATP1A1 is a highly specific phenotype because ER/Golgi-dependent protein transport of an integral membrane protein to the cell surface remained unaffected under these conditions. Along with reports suggesting the existence of unassembled ␣1-chains in plasma membranes (26), our findings indicate a direct role for ATP1A1 in the initiation of FGF2 membrane translocation at the inner leaflet of plasma membranes. This view is supported by biochemical experiments demonstrating a direct interaction with submicromolar affinity between the cytoplasmic domain of ATP1A1 and FGF2.…”

“…9, we propose ATP1A1 to be part of a chain of sequential interactions at the inner leaflet resulting in FGF2 oligomerization and membrane insertion. Intriguingly, unassembled ␣-chains have been proposed to exist in plasma membranes based on significantly faster turnover rates of ␤-versus ␣-chains (26). It is therefore an interesting working hypothesis that unassembled ␣1-chains (ATP1A1) play a role in unconventional secretion of FGF2 that is unrelated to their participation in the ␣ 2 ␤ 2 heterooligomeric complex that makes up a functional Na/K-ATPase.…”

Developmental changes in β-subunit composition of Na,K-ATPase in the Drosophila eye

“…Thus, the α-subunit in the microvilli of midpupal photoreceptors might occur without a β-subunit. We have shown recently that Na,K-ATPase α/β heterodimers in cultured vertebrate cells disassemble after transport to the plasma membrane and that both subunits are then subjected to different degradation processes leaving an excess of α-subunits in the plasma membrane (Yoshimura et al 2008). …”

Some Assembly Required: Putting the Epithelial Sodium Channel Together

“…that the ␣-and ␤-subunits of the Na ϩ /K ϩ -ATPase, which assembled in the ER, could dissociate in the plasma membrane and undergo differential degradation (32). The possibility of ENaC remodeling would likely depend on the ability of the channel to recycle.…”