Topology of the .alpha.-Subunit of Na,K-ATPase Based on Proteolysis. Lability of the Topological Organization

Abstract

Topology of the alpha-subunit of Na,K-ATPase has been analyzed utilizing proteolytic digestion. Evidence is presented for a model with 10 transmembrane segments and lability of the C-terminal domain (M7-M10). Using reconstituted proteoliposomes, inside-out oriented pumps were digested with trypsin at the cytoplasmic surface. Evidence was obtained for the M7/M8 pair and cytoplasmic splits between M8 and M9 and between M9 and M10. Because an extracellular split between M9 and M10 was also observed, using right-side-out oriented renal microsomes, we propose that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile. Using renal microsomes, extracellular digestion of the alpha-subunit by trypsin, chymotrypsin, or an endogenous protease has been observed, after incubation at 55 or at 45 degrees C with beta-mercaptoethanol (beta-ME) and n-butanol. Both perturbations inactivate enzyme activity. Rb ions protect against inactivation and digestion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymotrypsin cut between M7 and M8 and between M9 and M10, consistent with the 10-segment model. At 55 degrees C, the topological organization is altered, the M8/M9 connecting loop is exposed at the extracellular surface, and an additional split between M8 and M9 is observed. Extracellular digestion of the alpha-subunit is associated with digestion of the beta-subunit near the first extracellular S-S bridge. Rb ions protect the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interactions.

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

“…The presence of RbCl or KCl protected against the partial denaturation of both ␣ and ␤ subunits. Similar observations of K ϩ protection and the extrusion of M8 and M9 were made by others with pig kidney Na,KATPase (12). Some issues remained unaddressed by the experiments summarized in Fig.…”

“…3 Thus the gapped-BLAST alignment is consistent with the adoption of the same fold. Homologous C-terminal topology for the individual transmembrane hairpins of Na,K-ATPase is now supported by biochemical evidence (12,24). Cross-linking data suggests proximity of M1-M2 to M8 -10 in the Na,K-ATPase (25,26), and this can be accommodated in the model by the fact that M2 is adjacent to M9, and both have cysteine residues that could be the cause of the observed cross-link.…”

“…According to the data of Goldshleger et al (12), heating at 45°C with 1-butanol and ␤-mercaptoethanol exposes the extracellular L7-8 and L9 -10 loops to digestion. Heating at 50°C with dithiothreitol or at 55°C without it next exposes the intracellular L8 -9 loop at the extracellular surface (11,12).…”

“…According to the data of Goldshleger et al (12), heating at 45°C with 1-butanol and ␤-mercaptoethanol exposes the extracellular L7-8 and L9 -10 loops to digestion. Heating at 50°C with dithiothreitol or at 55°C without it next exposes the intracellular L8 -9 loop at the extracellular surface (11,12). We also observed (here and earlier) that the C terminus was exposed to the extracellular side, and since Goldshleger et al (12) did not, the extrusion of M10 and the C terminus might be the last step in the denaturation of the C-terminal portion of the protein.…”

“…Consequently much higher temperatures are required to denature the membrane domain. In this context, the observation that relatively mild heating (50°C in ␤-mercaptoethanol or 55°C without) caused a radical reorganization of part of the transmembrane domain of the Na,K-ATPase ␣ subunit while unfolding the ␤ subunit ectodomain was surprising (11,12). Here we have investigated further the consequences of heat denaturation of the Na,KATPase.…”