COLOCALIZATION OF RH POLYPEPTIDES AND THE AMINOPHOSPHOLIPID TRANSPORTER IN DILAUROYLPHOSPHATIDYLCHOLINE-INDUCED ERYTHROCYTE VESICLES

Cytoskeleton-free vesicles released from human red blood cells (RBC) t ransport exogenously supplied aminophospholipid analogues from the ves icle's outer to inner leaflet at rates comparable to those of normal R BC (Beleznay et al. (1993) Biochemistry 32, 3146-3152). Because polype ptides associated with the Rh blood group system have been implicated in the transbilayer movement of phosphatidylserine (PS), we investigat ed the relationship and co-localization of the aminophospholipid trans locase and Rh in dilauroylphosphatidylcholine-induced RBC vesicles. Th e transbilayer movement of fluorescent (NBD-PS) and photoactivatable ( I-125-N-3-PS) PS in RBC vesicles was ATP- and temperature-dependent. I nhibition of PS transport by sulfhydryl reagents could be accomplished by direct vesicle treatment or by treating RBC before vesiculation. I n the case of diamide- and pyridyldithioethylamine-mediated inhibition , NBD-PS transport could be restored by reduction with dithiothreitol, indicating that the movement of the PS transporter into the emerging vesicle was independent of the oxidative status of membrane sulfhydryl s. The presence of Rh polypeptides in the vesicles was verified by dir ect immunoprecipitation of isotopically-labeled Rh and semi-quantified by antibody adsorption assays. Similar to the movement of the PS tran sporter, localization of Rh polypeptides in the vesicle membrane was i ndependent of the red cell's oxidative status. These results show that the PS translocase and Rh-related proteins colocalize in RBC vesicles suggesting that these proteins may be members of a multicomponent com plex that plays a role in lipid movement and the generation of membran e lipid asymmetry.