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

Phospholemman (FXYD1), mainly expressed in heart and skeletal muscle, is a member of the FXYD protein family, which has been shown to decrease the apparent K

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase ␣1-␤1 and ␣1-␤2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K ؉ and Na ؉ affinities of ␣1-␤1 and ␣1-␤2 isozymes are different. Association of FXYD6 with Na,K-ATPase ␣1-␤1 isozymes slightly decreases their apparent K ؉ affinity and significantly decreases their apparent Na ؉ affinity. On the other hand, association with ␣1-␤2 isozymes increases their apparent K ؉ and Na ؉ affinity. The effects of FXYD6 on the apparent Na ؉ affinity of Na,KATPase and the voltage dependence of its K ؉ effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Six of 7 FXYD proteins have been shown to be tissue-specific modulators of Na,K-ATPase. In this study, we have identified two splice variants of human FXYD3, or Mat-8, in CaCo-2 cells. Short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation of CaCo-2 cells. Long human FXYD3 is mainly expressed in nondifferentiated cells and short human FXYD3 in differentiated cells and both FXYD3 variants can be co-immunoprecipitated with a Na,K-ATPase antibody. In contrast to mouse FXYD3, which has two transmembrane domains for lack of cleavage of the signal peptide, human FXYD3 has a cleavable signal peptide and adopts a type I topology. After co-expression in Xenopus oocytes, both human FXYD3 variants associate stably only with Na,K-ATPase isozymes but not with H,K-ATPase or Ca-ATPase. Similar to mouse FXYD3, short human FXYD3 decreases the apparent K ؉ and Na ؉ affinity of Na,K-ATPase over a large range of membrane potentials. On the other hand, long human FXYD3 decreases the apparent K ؉ affinity only at slightly negative and positive membrane potentials and increases the apparent Na ؉ affinity of Na,K-ATPase. Finally, both short and long human FXYD3 induce a hyperpolarization activated current, similar to that induced by mouse FXYD3. Thus, we have characterized two human FXYD3 isoforms that are differentially expressed in differentiated and non-differentiated cells and show different functional properties.

Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded ␤m-proteins in fish, amphibian, and avian species are ␤-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals ␤m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian ␤m acquired new functions as exemplified by regulation of TGF-␤-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-␤ signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins.ATP1B4 ͉ gene cooption ͉ skeletal muscle development ͉ TGF-␤ ͉ Smad7