Topological assessment of oatp1a1: a 12-transmembrane domain integral membrane protein with three N-linked carbohydrate chains

2008 ◽  
Vol 294 (4) ◽  
pp. G1052-G1059 ◽  
Author(s):  
Pijun Wang ◽  
Soichiro Hata ◽  
Yansen Xiao ◽  
John W. Murray ◽  
Allan W. Wolkoff
Keyword(s):  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Integral Membrane Protein ◽  
Glycosylation Site ◽  
Domain Model ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Glycosylation Sites

Organic anion transport protein 1a1 (oatp1a1), a prototypical member of the oatp family of highly homologous transport proteins, is expressed on the basolateral (sinusoidal) surface of rat hepatocytes. The organization of oatp1a1 within the plasma membrane has not been well defined, and computer-based models have predicted possible 12- as well as 10-transmembrane domain structures. Which of oatp1a1's four potential N-linked glycosylation sites are actually glycosylated and their influence on transport function have not been investigated in a mammalian system. In the present study, topology of oatp1a1 in the rat hepatocyte plasma membrane was examined by immunofluorescence analysis using an epitope-specific antibody designed to differentiate a 10- from a 12-transmembrane domain model. To map glycosylation sites, the asparagines at the each of the four N-linked glycosylation consensus sites were mutagenized to glutamines. Mutagenized oatp1a1 constructs were expressed in HeLa cells, and effects on protein expression and transport activity were assessed. These studies revealed that oatp1a1 is a 12-transmembrane-domain protein in which the second and fifth extracellular loops are glycosylated at asparagines 124, 135, and 492, whereas the potential glycosylation site at asparagine 62 is not utilized, consistent with its position in a transmembrane domain. Constructs in which more than one glycosylation site were eliminated had reduced transport activity but not necessarily reduced transporter expression. This was in accord with the finding that fully unglycosylated oatp1a1 was well expressed but located intracellularly with limited transport ability as a consequence of its reduced cell surface expression.

N-glycosylation controls functional activity of Oatp1, an organic anion transporter

2003 ◽  
Vol 285 (2) ◽  
pp. G371-G381 ◽  
Author(s):  
Thomas K. Lee ◽  
Albert S. Koh ◽  
Zhifeng Cui ◽  
Robert H. Pierce ◽  
Nazzareno Ballatori
Keyword(s):  
Plasma Membrane ◽  
Functional Activity ◽  
Plasma Membranes ◽  
Organic Anion ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Anion Transporter ◽  
Glycosylation Sites ◽  
Quadruple Mutant ◽  
Taurocholate Transport

Rat Oatp1 (Slc21a1) is an organic anion-transporting polypeptide believed to be an anion exchanger. To characterize its mechanism of transport, Oatp1 was expressed in Saccharomyces cerevisiae under control of the GAL1 promoter. Protein was present at high levels in isolated S. cerevisiae secretory vesicles but had minimal posttranslational modifications and failed to exhibit taurocholate transport activity. Apparent molecular mass ( M) of Oatp1 in yeast was similar to that of unmodified protein, ∼62 kDa, whereas in liver plasma membranes Oatp1 has an M of ∼85 kDa. To assess whether underglycosylation of Oatp1 in yeast suppressed functional activity, Oatp1 was expressed in Xenopus laevis oocytes with and without tunicamycin, a glycosylation inhibitor. With tunicamycin, M of Oatp1 decreased from ∼72 to ∼62 kDa and transport activity was nearly abolished. Mutations to four predicted N-glycosylation sites on Oatp1 (Asn to Asp at positions 62, 124, 135, and 492) revealed a cumulative effect on function of Oatp1, leading to total loss of taurocholate transport activity when all glycosylation sites were removed. M of the quadruple mutant was ∼ 62 kDa, confirming that these asparagine residues are sites of glycosylation in Oatp1. Relatively little of the quadruple mutant was able to reach the plasma membrane, and most remained in unidentified intracellular compartments. In contrast, two of the triple mutants tested (N62/124/135D and N124/135/492D) were present in the plasma membrane fraction yet exhibited minimal transport activity. These results demonstrate that both membrane targeting and functional activity of Oatp1 are controlled by the extent of N-glycosylation.

Plasma membrane delivery of the gastric H,K-ATPase: the role of β-subunit glycosylation

2003 ◽  
Vol 285 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
O. Vagin ◽  
S. Denevich ◽  
G. Sachs
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Glycosylation Site ◽  
Β Subunit ◽  
Wild Type ◽  
Α Subunit ◽  
Hek 293 ◽  
Glycosylation Sites ◽  
293 Cells

The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase β-subunit (YFP-β) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase α-subunit (CFP-α) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-α, wild-type YFP-β, and YFP-β mutants lacking one or two of the seven β-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-β resulted in the plasma membrane localization of the protein, whereas the expressed CFP-α was retained intracellularly. When coexpressed, both CFP-α and YFP-β were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-β prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the β-subunit are essential for the plasma membrane delivery of the β-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asn103), which is not conserved among the β-subunits from different species, is not critical for plasma delivery of the protein.

scholarly journals N-glycosylation requirements for the AT1a angiotensin II receptor delivery to the plasma membrane

1999 ◽  
Vol 339 (2) ◽  
pp. 397-405 ◽  
Author(s):  
Benoit DESLAURIERS ◽  
Cecilia PONCE ◽  
Colette LOMBARD ◽  
Renée LARGUIER ◽  
Jean-Claude BONNAFOUS ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Cellular Localization ◽  
Inositol Phosphate ◽  
Glycosylation Site ◽  
Receptor Expression ◽  
Site Directed Mutagenesis ◽  
Pharmacological Properties ◽  
Peptide Epitope ◽  
Glycosylation Sites

The purpose of this work was to investigate the role of N-glycosylation in the expression and pharmacological properties of the the rat AT1a angiotensin II (AII) receptor. Glycosylation-site suppression was carried out by site-directed mutagenesis (Asn → Gln) of Asn176 and Asn188 (located on the second extracellular loop) and by the removal of Asn4 at the N-terminal end combined with the replacement of the first four amino acids by a 10 amino acid peptide epitope (c-Myc). We generated seven possible N-glycosylation-site-defective mutants, all tagged at their C-terminal ends with the c-Myc epitope. This double-tagging strategy, associated with photoaffinity labelling, allowed evaluation of the molecular masses and immunocytochemical cellular localization of the various receptors transiently expressed in COS-7 cells. We showed that: (i) each of the three N-glycosylation sites are utilized in COS-7 cells; (ii) the mutant with three defective N-glycosylation sites was not (or was very inefficiently) expressed at the plasma membrane and accumulated inside the cell at the perinuclear zone; (iii) the preservation of two sites allowed normal receptor delivery to the plasma membrane, the presence of only Asn176 ensuring a behaviour similar to that of the wild-type receptor; and (iv) all expressed receptors displayed unchanged pharmacological properties (Kd for 125I-sarcosine1-AII; sarcosine1-AII-induced inositol phosphate production). These results demonstrate that N-glycosylation is required for the AT1 receptor expression. They are discussed in the light of current knowledge of membrane-protein maturation and future prospects of receptor overexpression for structural studies.

scholarly journals Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 580-586 ◽  
Author(s):  
EA Rutledge ◽  
BJ Root ◽  
JJ Lucas ◽  
CA Enns
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Transferrin Receptor ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Soluble Form ◽  
Protein Sequence Analysis ◽  
Wild Type ◽  
Human Transferrin Receptor ◽  
Intracellular Compartments

The transferrin receptor (TfR) is the plasma membrane protein responsible for the binding and internalization of the major iron- transport protein, transferrin. The function of the single O-linked oligosaccharide near the transmembrane domain of the TfR at amino acid Thr 104 is unknown. To elucidate the effect of the O-linked carbohydrate on TfR function, the oligosaccharide was eliminated by replacing Thr 104 with Asp and the mutated cDNA was expressed in a cell line lacking endogenous TfR. Elimination of the oligosaccharide at Thr 104 results in a form of the receptor that is susceptible to cleavage. A 78-kD soluble TfR that can bind transferrin is released into the growth medium. The intact mutant TfR is not grossly altered in its structure and does not differ significantly from the wild-type human receptor in many respects: (1) It shows the same distribution between the plasma membrane and intracellular compartments; (2) the binding constant for transferrin is similar to that of the wild-type TfR; and (3) it is not rapidly degraded. Protein-sequence analysis of the soluble form indicates that the sequence begins at amino acid 101 of the intact receptor. This is the same cleavage site reported for a soluble form of normal receptor found in human serum. Substitution of Gly, Glu, or Met at position 104 also results in increased cleavage of the TfR and suggests that elimination of the O-linked carbohydrate at position 104 enhances the susceptibility of TfR to cleavage and may mimic a naturally occurring process previously described as being related to erythropoiesis.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

scholarly journals Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

2009 ◽  
Vol 296 (3) ◽  
pp. C570-C582 ◽  
Author(s):  
Simone Leuthold ◽  
Bruno Hagenbuch ◽  
Nilufar Mohebbi ◽  
Carsten A. Wagner ◽  
Peter J. Meier ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Organic Anion ◽  
Extracellular Ph ◽  
Disulfonic Acid ◽  
Substrate Affinity ◽  
Xenopus Laevis Oocytes ◽  
Transport Activity ◽  
Substrate Transport ◽  
Mammalian Cell Lines ◽  
Ph Dependency

Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.

scholarly journals Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor

2005 ◽  
Vol 390 (1) ◽  
pp. 367-376 ◽  
Author(s):  
Pascal M. Lanctot ◽  
Patrice C. Leclerc ◽  
Martin Clément ◽  
Mannix Auger-Messier ◽  
Emanuel Escher ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Surface ◽  
Functional Expression ◽  
Surface Expression ◽  
Glycosylation Site ◽  
At1 Receptor ◽  
Cell Surface Expression ◽  
Receptor Subtype ◽  
Angiotensin Ii Receptor ◽  
Glycosylation Sites

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT1 receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.

scholarly journals Cleavage of membrane secretory component to soluble secretory component occurs on the cell surface of rat hepatocyte monolayers.

1987 ◽  
Vol 104 (6) ◽  
pp. 1725-1733 ◽  
Author(s):  
L S Musil ◽  
J U Baenziger
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Rat Hepatocytes ◽  
Integral Membrane Protein ◽  
Secretory Component ◽  
Soluble Form ◽  
Membrane Domain ◽  
Transcellular Transport ◽  
Cultured Rat Hepatocytes ◽  
Plasma Membrane Domain

Rat liver secretory component is synthesized as an integral membrane protein (mSC) and cleaved to an 80-kD soluble form (fSC) sometime during transcellular transport from the sinusoidal to the bile canalicular plasma membrane domain of hepatocytes. We have used 24-h monolayer cultures of rat hepatocytes to characterize the conversion of mSC to fSC. Cleavage of mSC in cultured hepatocytes is inhibited by the thiol protease inhibitors leupeptin, antipain, and E-64, but not by other inhibitors, including disopropylfluorophosphate, pepstatin, N-ethylmalemide, p-chloromercuribenzoic acid, and chloroquine. Leupeptin-mediated inhibition of cleavage is concentration dependent and reversible. In the presence or absence of leupeptin, only 10-20% of mSC is accessible at the cell surface. To characterize the behavior of surface as opposed to intracellular mSC, cell surface mSC was labeled with 125I by lactoperoxidase-catalyzed iodination at 4 degrees C. Cell surface 125I-mSC was converted to extracellular fSC at 4 degrees C in the absence of detectable internalization. Cleavage was inhibited by leupeptin and by anti-secretory component antiserum. Cleavage also occurred at 4 degrees C after cell disruption. In contrast, 125I-mSC that had been internalized from the cell surface was not converted to fSC at 4 degrees C in either intact or disrupted cells. Hepatocytes metabolically labeled with [35S]cys also released small quantities of fSC into the medium at 4 degrees C. The properties of fSC production indicate that cleavage occurs on the surface of cultured rat hepatocytes and not intracellularly. Other features of the cleavage reaction suggest that the mSC-cleaving protease is segregated from the majority of cell surface mSC, possibly within a specialized plasma membrane domain.

scholarly journals Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

2003 ◽  
Vol 14 (3) ◽  
pp. 1268-1278 ◽  
Author(s):  
Laurence Fayadat ◽  
Ron R. Kopito
Keyword(s):  
Quality Control ◽  
Plasma Membrane ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Proteasome Inhibitors ◽  
Integral Membrane Protein ◽  
Type I ◽  
Α Subunit ◽  
Influenza Hemagglutinin

To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document