DEMONSTRATION OF EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS (HENT1 AND HENT2) IN NUCLEAR ENVELOPES OF CULTURED HUMAN CHORIOCARCINOMA (BEWO) CELLS BY FUNCTIONAL RECONSTITUTION IN PROTEOLIPOSOMES

The equilibrative nucleoside transporters (ENTs) are a newly recognize d family of membrane proteins of which hENT1 is the nitrobenzylmercapt opurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-inse nsitive (ei) transporter of human cells. BeWo cells exhibit large numb ers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we h ave demonstrated that proliferating BeWo cells possess (i) mRNA encodi ng hENT1 and hENT2 and (ii) hENT1-specific immuno-epitopes. We examine d NBMPR binding and its inhibition of uridine transport in various BeW o membrane fractions and proteoliposomes derived therefrom to determin e if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fraction s enriched 5-fold in plasma membranes relative to postnuclear supernat ants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites wi th affinities similar to those of enriched plasma membranes (K-d = 0.4 -0.9 nM). When proteoliposomes were made from octyl glucoside-solubili zed membranes, high affinity NBMPR-binding sites were not only observe d in crude membrane preparations and plasma membrane-enriched fraction s but also in nuclear envelope fractions. Proteoliposomes prepared fro m either unfractionated membranes or nuclear envelopes exhibited both hENT1-mediated (82-85%) and hENT2-mediated (15-18%) transport of [H-3] uridine. These results provided evidence for the presence of functiona l es and ei transporters in nuclear membranes and endoplasmic reticulu m, suggesting that hENT1 and hENT2 may function in the translocation o f nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.