Molecular Cloning and Immunological Characterization of the / Polypeptide , a Small Protein Associated with the Na , K-ATPase

The 3' subunit of the Na,K-ATPase is a small membrane protein that copurifies with the ct and subunits of the enzyme. Strong evidence that the 3' subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the 3' subunit from several species. The 3' subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presenc.e of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the 3' subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. 3'-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the 3' subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the ot subunit. In addition, coimmunoprecipitation of the or, B and 3' subunits demonstrate specific association of the subunits. These results are consistent with the notion that the 3' subunit is specifically associated with and may be an important component of the Na,K-ATPase. T HE Na,K-ATPase is a membrane protein that uses the energy from the hydrolysis of ATP to transport Na and K across the plasma membrane. The enzyme consists of at least two polypeptides, a catalytic ct subunit of *110 kD and a smaller glycosylated/3 subunit of '~55 kD. The ot subunit contains the binding site for ATP and the cardiac glycoside inhibitor ouabain; it is phosphorylated and undergoes ligand-dependent conformation changes accompanying the binding and translocation of cations across the plasma membrane. All catalytic functions have been assigned to the t~ subunit, however, the/~ subunit may play an essential role in either stabilization, maturation, or enzymatic activity of functional Na,K-ATPase molecules (Gerring, 1991). The amino acid sequences of the ct and/~ subunits of the Na,K-ATpase from several species have been determined from their cDNAs (reviewed in Lingrel et al., 1990; Mercer et al., 1989). Distinct forms of the rat Na,K-ATPase have been described which can be distinguished by SDS-PAGE (Sweadner, 1979), antigenic determinants (Sweadner and Gilkeson, 1985), and differences in affinity for ouabain (Sweadner, 1979). These differences have been ascribed to the presence of two isoforms of the o~ subunit (or and ct[+]). The cDNAs for the or(+) isoform (termed ct2) and an ot subunit previously unidentified, designated or3, have been isolated and sequenced (Shull et al., 1986; Takeyasu et al., 1990). In addition to the ot isoforms, a putative/~ subunit isoform cDNA, termed/~2, has been identified in mammals (Martin-Vasallo et al., 1989). In glia cells this polypeptide functions as an adhesion molecule and is associated with the or2 subunit of the Na,K-ATPase (Gloor et al., 1990). Characterization of a, c~ isoform, and B mRNAs has demonstrated that the subunits are expressed in a tissue-specific and developmentally regulated fashion (Mercer et al., 1986; Young and Lingrel, 1987; Schneider et al., 1988; Orlowski and Lingrel, 1988; Martin-Vasallo et al., 1989). Thus, the diversity in subunits and their differential expression and regulation suggests that the Na,K-ATPase may have several specialized functional roles. Besides the other subunits, a small peptide of •10 kD, termed the 3, subunit (previously "proteolipid component"), has also been identified in purified preparations of the enzyme. Strong evidence that the 3, subunit is a component of the Na,K-ATPase and not a contaminant of purification has come from studies of the cardiac glycoside binding site. These studies have shown that the binding site consists of not only the t~ and/3 subunits, but the ~/subunit as well (Forbush et al., 1978; Rogers and Lazdunski, 1979; Lowndes et al., 1984). For example, the c~ and 3' subunlts are labeled by photoaffinity compounds with approximately equal probability when the reactive groups are 23-24 ~, from the lactone ring of the cardiotonic steroid (Forbush, 1983). Although small hydrophobic proteins are associated with the H-ATPase of mitochondria (Blondin, 1979), bacteria (Sierra and Tza© The Rockefeller University Press, 0021-9525/93/05/579/8 $2.00 The Journal of Cell Biology, Volume 121, Number 3, May 1993 579-586 579 on M ay 1, 2017 D ow nladed fom Published May 1, 1993