Oligomerization and maturation of Na,K-ATPase: functional interaction of the cytoplasmic NH2 terminus of the beta subunit with the alpha subunit.

Abstract

Abstract. Subunit assembly plays an essential role in the maturation of oligomeric proteins. In this study, we have characterized the main structural and functional consequences of the assembly of a and [3 subunits of Na,K-ATPase. Xenopus oocytes injected with c~ and/or 13 cRNA were treated with brefeldin A, which permitted the accumulation of individual subunits or a-[3 complexes in the ER. Only ot subunits that are associated with 13 subunits become resistant to trypsin digestion and cellular degradation. Similarly, assembly with 13 subunits is necessary and probably sufficient for the catalytic et subunit to acquire its main functional properties at the level of the ER, namely the ability to adopt different ligand-dependent conformations and to hydrolyze ATP in an Na ÷-and K÷-dependent, ouabaininhibitable fashion. Not only the a but also the [3 subunit undergoes a structural change after assembly, which results in a global increase in its protease resistance. Furthermore, extensive and controlled proteolysis assays on wild-type and NHz-terminally modified 13 subunits revealed a K+-dependent interaction of the cytoplasmic NH2 terminus of the [3 subunit with the subunit, which is likely to be involved in the modulation of the K÷-activation of the Na,K-pump transport activity. Thus, we conclude that the ER assembly process not only establishes the basic structural interactions between individual subunits, which are required for the maturation of oligomeric proteins, but also distinct, functional interactions, which are involved in the regulation of functional properties of mature proteins.M ANY plasma membrane and secretory proteins are oligomeric. The subunits of these proteins are synthesized independenly of each other and are inserted into the ER membrane or the lumen during their synthesis. They are subjected to cotranslational modifications, fold, and then oligomerize. Once correctly assembled, the proteins leave the ER and are targeted to their final cellular site of action. Misfolded or unassembled subunits are retained in the ER and are degraded (15). Although it is increasingly clear that the oligomeric state controls the proper function of the protein, little is known about the nature of interactions that are involved in subunit assembly and about the structural and functional consequences of subunit oligomerization. In this study, we investigate several aspects of this question by analyzing the oligomerization and the functional maturation of Na,KATPase.The ubiquitous Na,K-ATPase is responsible for the maintenance of the sodium and potassium gradients between the intra-and extracellular milieu. The enzyme is composed of two heterologous subunits. The ct subunit is a

Endoplasmic Reticulum Quality Control of Oligomeric Membrane Proteins: Topogenic Determinants Involved in the Degradation of the Unassembled Na,K-ATPase α Subunit and in Its Stabilization by β Subunit Assembly

“…In contrast, alanine mutants affected in the central region of the Val-890 -Val-916 domain encompassing Asp-902 up to Tyr-910 were degraded during a 48-h chase period even in the presence of ␤ subunits ( Figure 6B, lanes 11-18). In agreement, ␤ subunits that were coexpressed with these assembly-incompetent ␣-mutants remained in their core glycosylated ER form and were slowly degraded ( Figure 6C, lanes 11-18), as expected for unassembled ␤ subunits (Geering et al, 1996). These results indicate that the previously reported 903 SYGQ 906 motif of ␣-␤ interaction (Colonna et al, 1997) is important but not sufficient for complete stabilization of ␣ subunits by the ␤ subunit.…”

“…Na,K-ATPase belongs to the P-type ATPase superfamily of cation transporters and is a ubiquitous plasma membrane enzyme responsible for intra-and extracellular Na and K homeostasis (for review, see Horisberger, 1994). Among the P-type ATPases, only the Na,K-and H,KATPase ␣ subunits require assembly with a partner ␤ subunit in the ER to become stably expressed, functionally active, and competent for intracellular transport (Geering et al, 1996;Beggah et al, 1999). Similar to most P-type ATPases, the ␣ subunits of Na,K-and H,K-ATPases have 10 transmembrane segments and a large central, cytoplasmic loop and expose the N and the C termini to the cytoplasmic side.…”

Mutational analysis of the Lem3p-Dnf1p putative phospholipid-translocating P-type ATPase reveals novel regulatory roles for Lem3p and a carboxyl-terminal region of Dnf1p independent of the phospholipid-translocating activity of Dnf1p in yeast

“…The β-subunit is likely required not only for transport out of the ER, but also for regulation of the Na + , K + -pump transport activity of the mature complex [27]. Interestingly, the lem3(R316W) mutant, which displayed normal polarized localization of Dnf1p-EGFP, was not impaired in its uptake of NBD-PE and -PC, suggesting that Lem3p(R316W) is defective in harnessing the putative APLT activity of Dnf1p for intracellular functions shared by Cdc50p-Drs2p.…”

Structural and Functional Interaction Sites between Na,K-ATPase and FXYD Proteins

“…The construction of the chimera ␣1-AL1 containing Met 1 to Arg 941 of the human Na,K-ATPase ␣1 subunit and Asn 960 to Tyr 1039 of the human non-gastric H,K-ATPase ␣ subunit (AL1) has been described previously (22) Protein Expression in Xenopus Oocytes and Metabolic LabelingcRNAs were obtained by in vitro transcription (23). Stage V-VI Xenopus oocytes were obtained as described (24). Oocytes were injected with cRNAs coding for wild type or mutant rat Na,K-ATPase ␣1 subunits (10 ng/oocyte) or for AL1 (10 ng/oocyte) (25) or ␣1-AL1 (10 ng/oocyte) together with cRNAs for the rat Na,K-ATPase ␤1 subunit (1 ng/oocyte) or rabbit gastric H,K-ATPase ␤ subunit (1 ng/oocyte) (kindly provided by G. Sachs) with or without cRNAs coding for human FXYD2a (11), rat FXYD4 (11), or mouse FXYD7 (2 ng) (17).…”

“…Oocytes were incubated in modified Barth's solution in the presence of 0.8 -1 mCi/ml [ 35 S]methionine (Easy Tag Express [ 35 S] protein labeling kit, PerkinElmer Life Sciences) at 19°C and subjected to a 6-h pulse and to 24-, 48-, and 72-h chase periods in modified Barth's solution containing 10 mM cold methionine. Microsomes were prepared after each chase period as described previously (24).…”