Abstract
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 immunoepitopes. 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 (Kd = 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 [3H]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.
Highlights
Multiple nucleoside transport (NT)1-mediated processes exist in mammalian cells and are divided into two groups, depending on whether they are equilibrative or concentrative in nature
Since hENT1 and hENT2 were initially identified by molecular cloning of a placental cDNA library [10, 16] and the BeWo cell line is of placental origin [18], BeWo RNA was examined for evidence of expression of hENT1 and hENT2 mRNA
These results suggested that the es and ei nucleoside transport activities of BeWo cells were mediated by the hENT1 and hENT2 gene products
Summary
Materials—[G-3H]NBMPR (22.5 Ci/mmol) and [5,6-3H]uridine (40 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA). [14C]Cholesteryl oleate (0.1 mCi/ml) was obtained from Amersham Canada Ltd. (Oakville, Canada). Proteoliposomes (ϳ10 g of protein/ml final concentration), and intact nuclei and nuclear membranes (at ϳ40 g of protein/ml) were incubated for 45 min at room temperature with a range of concentrations (0.24 –24 nM) of [3H]NBMPR in either the absence (total binding) or presence (nonspecific binding) of 10 M NBMPR (1-ml final assay volume). Estimates of zero time uptake values were obtained by measuring the uptake of [3H]uridine at ϳ2 s in the presence of ice-cold solutions containing 10 mM adenosine, 10 M dipyridamole, and 10 M NBTGR to inhibit all transporter-mediated influx. Flow Cytometry—Nuclei (obtained by cell disruption as described above) were collected by centrifugation (5 min, 500 ϫ g), washed twice in PBS, and resuspended at a density of 106 nuclei/ml. The specificity of the hENT1-(55– 64) antibodies was assessed by comparing fluorescence intensity of hENT1-containing cells in the presence and absence of excess hENT1-(55– 64)
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