The fifth cytoplasmic domain of Na+/K+-ATPase mediates its interaction with acetylated tubulin.
Zampar, G.G., Carbajal, A., Chesta, M.E., Díaz, N.M., Chanaday, N.L., Casale, C.H. and Arce, C.A.
CIQUIBIC (Conicet), Depto. de Química Biológica, Fac. de Ciencias Químicas, UNC
E-mail: gzampar@mail.fcq.unc.edu.ar
Na+/K+-ATPase is an integral membrane protein that transports 3 Na+ out of the cell in exchange for 2 K+ by using the energy of ATP hydrolysis. We had found that this enzyme interacts with acetylated tubulin inhibiting its catalytic activity. In the present work we demonstrate that this interaction is mediated by the fifth cytoplasmic domain (CD5) of the alpha subunit of Na+/K+-ATPase. Three observations support this conclusion: 1) A 17-amino acid peptide corresponding to the sequence of CD5 associates with tubulin in pull-down assays; 2) retained tubulin is mainly (if not exclusively) of the acetylated isotype; and 3) the interaction is inhibited by solubilized membranes that contain full-length Na+/K+-ATPase. Furthermore, we developed an antibody specific to CD5 and performed cross-linking experiments between CD5 and tubulin in order to determine whether the interaction is direct (i.e. without any intermediary compound). After the covalent cross-linking of CD5 with the tubulin preparation and subsequent SDS-PAGE, we found that CD5 migrated in a single band located slightly above the acetylated tubulin band. We also characterized the cross-linked aduct by acetic acid/urea-PAGE confirming our results. Taken together, these evidences strongly support the idea that CD5 mediates the interaction between Na+/K+-ATPase and acetylated tubulin in a direct manner.
