The transport modifier RS1 is localized at the inner side of the plasma membrane and changes membrane capacitance

Previously we cloned membrane associated (M-r 62 000-67 000) polypeptides f rom pig (pRS1), rabbit (rbRS1) and man (hRS1)which modified transport activ ities that were expressed in Xenopus laevis oocytes by the Na+-D-glucose co transporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier inmunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After su bcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was asso ciated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RSI is l ocalized at the inner side of the plasma membrane. Western blots with plasm a membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V-max of expressed glucose transport. Measurements of me mbrane capacitance and electron microscopic inspection showed that the expr ession of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that assoc iates with the plasma membrane. Overexpression of RS1 may effect the incorp oration and/or retrieval of transporters into the plasma membrane. (C) 2000 Elsevier Science B.V. All rights reserved.