REGULATION OF PHOSPHOLIPID TRANSLOCATION IN THE PLASMA MEMBRANE BY TUBULIN

TAMARA MUHLBERGER; MELISA BALACH; NOELIA MONESTEROLO; CESAR CASALE; ALEXIS N. CAMPETELLI

Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

The asymmetrical distribution of phospholipids in biological membranesplays major physiological roles at the cellular level. Thisasymmetry is generated and kept by transporters. Among them, thelipid flippases belongs to the family of P4-ATPases. Some P-ATPases,such as NKA and PMCA, are regulated by tubulin. In erythrocytesfrom hypertensive and diabetic subjects, tubulin is increasedin the plasma membrane (PM) and as a consequence, the NKA andPMCA activity results partially inhibited. The objective of this workwas to evaluate the putative inhibition of flippases by tubulin, bothin vitro and in vivo. Here, we used Inside-Out vesicles (IOVs) fromerythrocyte ghosts and the NBD labeled phosphathydilserine analogto test our hypothesis. When IOVs were incubated with purified tubulin,the NBD-PS translocation rate was decreased by 40% whencompared with controls without tubulin, suggesting that tubulin inhibitthe flippase activity in vitro. For the in vivo assay, erythrocyteswere loaded with NBD-PS and the rate of NBD-PS translocationwas assayed by flow cytometry. The results showed that the NBDPStranslocation rate is 20% slower in hypertensive and diabeticerythrocytes when compared with the control (erythrocytes from normalsubjects). In addition, when tubulin levels are pharmacologicallymodified in the PM of the erythropoietic K562 cells or erythrocytes,the percentage of NBD-PS translocated into the cell decreases intaxol-treated and in starved cells (conditions that promotes the associationof tubulin to the PM) and the opposite effect was observedin cells treated with nocodazol (a condition that removes tubulin fromthe PM). These results are consistent with those obtained in in vitroexperiments. Finally, we are currently performing pull down experimentsin order to test the putative interaction between ATP11C, themain flippase in erythrocytes and tubulin. These experiments willbring some light to understand this inhibition mechanism.