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 recognized family of membrane proteins of which hENT1 is the nitrobenzylmercaptopurine ribonucleoside (NBMPR)-sensitive (es) and hENT2 the NBMPR-insensitive (ei) transporter of human cells. BeWo cells exhibit large numbers (>10(7)/cell) of NBMPR-binding sites and high es and ei nucleoside transport activities relative to other cell types. In this work, we have demonstrated that proliferating BeWo cells possess (i) mRNA encoding hENT1 and hENT2 and (ii) hENT1-specific immuno-epitopes. We examined NBMPR binding and its inhibition of uridine transport in various BeWo membrane fractions and proteoliposomes derived therefrom to determine if NBMPR binding to intracellular membranes represented interaction with functional es transporters. Unfractionated membranes and fractions enriched 5-fold in plasma membranes relative to postnuclear supernatants exhibited high NBMPR binding activity. Intact nuclei and nuclear envelopes also exhibited abundant quantities of NBMPR-binding sites with affinities similar to those of enriched plasma membranes (K-d = 0.4-0.9 nM). When proteoliposomes were made from octyl glucoside-solubilized membranes, high affinity NBMPR-binding sites were not only observed in crude membrane preparations and plasma membrane-enriched fractions but also in nuclear envelope fractions. Proteoliposomes prepared from 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 functional es and ei transporters in nuclear membranes and endoplasmic reticulum, suggesting that hENT1 and hENT2 may function in the translocation of nucleosides between the cytosol and the luminal compartments of one or both of these membrane types.