| Abstract
| - The role of N-linked glycosylation of β-subunits in the functional properties of the oligomeric P-type ATPases Na,K- and H,K-ATPase has been examined by expressing glycosylation-deficient Asn-to-Gln β-variants in Xenopus oocytes. For both ATPases, the absence of the huge N-linked oligosaccharide moiety on the β-subunit does not affect α/β coassembly, plasma membrane delivery or functional activity of the holoenzyme. Whereas this is in line with several previous glycosylation studies on Na,K-ATPase, this is the first report showing that the cell surface delivery and enzymatic activity of the gastric H,K-ATPase is unaffected by the lack of N-linked glycosylation. Sulfhydryl-specific labeling of introduced cysteine reporter sites with the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes enabled us to further investigate potential effects of the N-glycans on more subtle enzymatic properties, like the distribution between E1P/E2P states of the catalytic cycle and the kinetics of the E1P/E2P conformational transition under presteady state conditions. For both Na,K-ATPase and H,K-ATPase, we observed differences in neither the voltage-dependent E1P/E2P ratio nor the kinetics of the E1P/E2P transition between holoenzymes comprising glycosylated and glycosylation-deficient β-subunits. We conclude that the N-linked glycans on these essential accessory subunits of oligomeric P-type ATPases are dispensable for proper folding, membrane stabilization of the α-subunit and transport function itself. Glycosylation is rather important for other cellular functions not relevant in the oocyte expression system, such as intercellular interactions or basolateral versus apical targeting in polarized cells, as demonstrated in other expression systems.
|
