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

Abstract

Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter 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 immunohistochemical 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 subcellular 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 associated 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 RS1 is localized at the inner side of the plasma membrane. Western blots with plasma 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 membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.

The Plasma Membrane-associated Protein RS1 Decreases Transcription of the Transporter SGLT1 in Confluent LLC-PK1 Cells

“…For SDSpolyacrylamide gel electrophoresis, protein samples were incubated for 1 h at 37°C in 50 mM Na 2 HPO 4 , pH 6.8, 4 M urea, 0.25 M ␤-mercaptoethanol, 1% (w/v) SDS, and 0.0005% (w/v) bromphenol blue. If immunodetection of pRS1 was intended, the SDS-treated protein samples were subsequently acetylated (29). After SDS-polyacrylamide gel electrophoresis performed according to Laemmli (41), the proteins were electrophoretically transferred to nitrocellulose membranes by semidry blotting (42).…”

“…For antibody reaction, the blots were incubated for 2 h at 25°C with affinity-purified polyclonal rabbit antibodies against porcine SGLT1 (pSGLT1-ab) or porcine RS1 (pRS1-ab) that were diluted 1:10 or 1:2500 in blocking buffer, respectively. The antibodies have been described earlier (29,43). pSGLT1-ab are raised against the amino acids 525-542 of porcine SGLT1 and affinity-purified on the antigenic peptide fixed to Sepharose, whereas pRS1-ab was raised against recombinant pRS1 protein expressed in E. coli and affinity-purified on the recombinant protein fixed to enzyme-linked immunosorbent assay plates.…”

“…Previously, it has been described (16 -19) that the expression of SGLT1 in LLC-PK 1 cells is up-regulated after confluence and repressed during cultivation in the presence of high D-glucose concentrations in the medium (25 versus 5 mM). As co-expression experiments of RS1 with SGLT1 in Xenopus oocytes suggested species-dependent up-or down-regulation of SGLT1 by RS1 (26,29,45), we investigated whether any alterations of pRS1 expression occur during the course of SGLT1 regulation by confluence and glucose concentrations. By confirming previous data, we observed that phlorizin-inhibitable uptake into subconfluent LLC-PK 1 cells was very low and not significantly different after cultivation in low versus high Dglucose.…”

“…Based on these findings, we put forward the hypothesis that RS1 is a subunit of SGLT1. This hypothesis was recently abandoned because we found that RS1 is actually localized at the intracellular face of the plasma membrane and that overexpression of human RS1 in Xenopus oocytes not only decreased the expression of human SGLT1 but also that of the human organic cation transporter hOCT2 (28,29). In the present study, LLC-PK 1 cells were used to investigate the putative role of RS1 in the regulation of SGLT1 ex-pression and activity by cell confluence and extracellular glucose concentration.…”

“…2a). pSGLT1 was detected with the affinity-purified, subtype-specific antibody pSGLT1-ab (43) and pRS1 with the affinitypurified antibody pRS1-ab that was raised against recombinant pRS1 protein (29). In Western blots on purified brushborder membranes from pig kidney, pSGLT1-ab binds to a polypeptide band at about 75 kDa, whereas pRS1-ab detects a polypeptide band at about 100 kDa (see control in Fig.…”