ANCHORAGE OF MICROTUBULES TO PLASMA MEMBRANE THROUGH INTERACTION WITH Na,K-ATPase

ZAMPAR G.G.; CARBAJAL A.; SANTANDER V.; CHANADAY N.; DÍAZ N.; CASALE C.H.; ARCE C.A.

Mar del Plata, Buenos Aires, Argentina

Congreso; SAIB; 2007

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 previously found that this enzyme interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we found that, during molecular filtration chromatography, the complex eluted at volume corresponding to higher MW than those of each protein component alone. We determined that tubulin forming part of microtubules can interact with Na,K-ATPase because: 1) native microtubules depleted of membranes  have associated Na,K-ATPase and 2) microtubules assembled from purified tubulin were able to associate Na,K-ATPase  when incubated with a detergent-solubilized membrane preparation. Furthermore, we tentatively identified CD5 (cytoplasmic domain 5) as the cytoplasmic fragment of Na,K-ATPase capable of interacting with acetylated tubulin. We tested the six cytoplasmic fragments and only CD5 bound to acetylated tubulin. Two of the peptides were obtained commercially and four as recombinant proteins. Taken together, our results are consistent with the idea that Na,K-ATPase may act as an anchorage point of microtubules on plasma membrane.