scholarly journals Intracellular Trafficking of Variant Chicken Kidney Ae1 Anion Exchangers

1999 ◽  
Vol 147 (6) ◽  
pp. 1237-1248 ◽  
Author(s):  
Tracy L. Adair-Kirk ◽  
Kathleen H. Cox ◽  
John V. Cox
Keyword(s):  
Amino Acids ◽  
Actin Cytoskeleton ◽  
Anion Exchanger ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Critical Role ◽  
Anion Exchangers ◽  
Tyrosine Residues ◽  
Chicken Kidney

The variant chicken kidney AE1 anion exchangers differ only at the NH2 terminus of their cytoplasmic domains. Transfection studies have indicated that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK kidney epithelial cells, while the AE1-3 variant, which lacks the NH2-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. Mutagenesis studies have shown that the basolateral accumulation of AE1-4 is dependent upon two tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of MDCK cells. Pulse–chase studies have shown that after delivery to the cell surface, newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. This Golgi recycling activity is dependent upon the same cytoplasmic tyrosine residues that are required for the basolateral sorting of this variant transporter. Furthermore, mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton and are rapidly turned over. These studies, which represent the first description of tyrosine-dependent cytoplasmic sorting signal for a type III membrane protein, have suggested a critical role for the actin cytoskeleton in regulating AE1 anion exchanger localization and stability in this epithelial cell type.

Variant chicken AE1 anion exchangers possess divergent NH(2)-terminal cytoplasmic domains

1995 ◽  
Vol 268 (3) ◽  
pp. F503-F513 ◽  
Author(s):  
K. H. Cox ◽  
J. V. Cox
Keyword(s):  
Amino Acids ◽  
Anion Exchanger ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Primary Transcript ◽  
Anion Exchangers ◽  
Transcriptional Initiation ◽  
Terminal Amino ◽  
Chicken Kidney

Immunoblotting analyses have demonstrated that antibodies specific for the chicken erythroid AE1 anion exchanger recognize multiple polypeptides ranging in size from approximately 95 to 112 kDa in chicken kidney. To determine the origin of this diversity, we have cloned and characterized the kidney AE1 anion exchangers. These studies have shown that the kidney AE1 polypeptides are encoded by at least three transcripts, AE1-3, AE1-4, and AE1-5, which differ from the erythroid AE1-1 and AE1-2 transcripts in the sequences present at their 5'-ends. The AE1-3 and AE1-5 transcripts encode predicted polypeptides of approximately 94 kDa, which are identical to the erythroid AE1-1 anion exchanger except for the absence of the 78 NH(2)-terminal amino acids of the AE1-1 polypeptide. In contrast, the AE1-4 transcript encodes a predicted polypeptide of approximately 101 kDa, whose 21 NH(2)-terminal amino acids are unique. Characterization of the AE1 cDNAs has suggested that the AE1-3 and AE1-4 transcripts are generated by alternative splicing of a single primary transcript, while DNA blotting analyses have shown that the putative transcription initiation sites of the variant AE1-4 and AE1-5 transcripts lie several kilobases downstream of the transcription initiation sites of the erythroid AE1-1 and AE1-2 transcripts. These results suggest that the pattern of accumulation of the variant kidney AE1 anion exchangers is regulated by a complex pattern of alternative transcriptional initiation and differential RNA splicing.

scholarly journals Polarized Distribution of IQGAP Proteins in Gastric Parietal Cells and Their Roles in Regulated Epithelial Cell Secretion

2003 ◽  
Vol 14 (3) ◽  
pp. 1097-1108 ◽  
Author(s):  
Rihong Zhou ◽  
Zhen Guo ◽  
Charles Watson ◽  
Emily Chen ◽  
Rong Kong ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Actin Cytoskeleton ◽  
Acid Secretion ◽  
Rho Gtpase ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Parietal Cells ◽  
Actin Dynamics ◽  
Cell Secretion ◽  
Gastric Parietal Cells

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

scholarly journals Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

2005 ◽  
Vol 168 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Doris Meder ◽  
Anna Shevchenko ◽  
Kai Simons ◽  
Joachim Füllekrug
Keyword(s):  
Free Surface ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Binding Motif ◽  
Membrane Domains ◽  
Regulatory Factor ◽  
Pdz Protein

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)–binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

2007 ◽  
Vol 292 (4) ◽  
pp. G1079-G1088 ◽  
Author(s):  
Janet E. Simpson ◽  
Clifford W. Schweinfest ◽  
Gary E. Shull ◽  
Lara R. Gawenis ◽  
Nancy M. Walker ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Duodenal Mucosa ◽  
Transport Processes ◽  
Wild Type ◽  
Rt Pcr ◽  
Targeted Deletion ◽  
Anion Transporter ◽  
Dependent Transport

Basal HCO3− secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl−/HCO3− exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl−/HCO3− exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl−/HCO3− exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO42−/HCO3− exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pHi) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO2/HCO3− or during methazolamide treatment. Further experiments under physiological conditions indicated active pHi compensation in the PAT-1(−) villous epithelium by combined activities of Na+/H+ exchanger 1 and Cl−-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl−/HCO3− and SO42−/HCO3− exchange across the apical membrane and 2) PAT-1 plays a role in pHi regulation in the upper villous epithelium of the murine duodenum.

scholarly journals A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells

1993 ◽  
Vol 289 (1) ◽  
pp. 263-268 ◽  
Author(s):  
S O Rosenberg ◽  
T Fadil ◽  
V L Schuster
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ionic Diffusion ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Disulphonic Acid ◽  
Component Carrier ◽  
Darby Canine Kidney ◽  
Canine Kidney

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

scholarly journals Fluid transport in a cultured cell model of kidney epithelial cyst enlargement.

1992 ◽  
Vol 2 (7) ◽  
pp. 1208-1218
Author(s):  
G A Tanner ◽  
M R Maxwell ◽  
J A McAteer
Keyword(s):  
Fluid Transport ◽  
Cell Model ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Critical Role ◽  
Collagen Gel ◽  
Fluid Secretion ◽  
Cyst Fluid ◽  
Disulfonic Acid ◽  
Renal Cystic Disease

Madin-Darby canine kidney (MDCK) cells, when seeded into collagen gel, from polarized, spherical, epithelial cysts, which grow by a process involving fluid secretion and cell proliferation. These cysts are a useful model for understanding the dynamics of cyst enlargement in renal cystic disease. The hypothesis that MDCK cyst fluid secretion depends upon chloride secretion was tested, and a cell model for this process is presented here. Lumen and epithelial cell volumes were measured by video microscopy in acute experiments. Fluid absorption (-0.073 +/- 0.007 microliters.h-1.cm-2; N = 8) was observed when cysts were superfused with unsupplemented Dublecco's modified Eagle's medium at 36 to 37 degrees C. Fluid secretion (0.221 +/- 0.0016 microliters.h-1.cm-2; N = 25) was seen when 1 mM dibutyryl cAMP plus 0.1 mM 3-isobutyl-1-methylxanthine were added to the superfusate. cAMP-induced fluid secretion was significantly inhibited by basolateral 1 mM ouabain, 0.1 mM furosemide, or 1 mM amiloride. It was not significantly affected by 1 mM chlorothiazide, 0.01 mM bumetanide, or 0.1 mM acetazolamide in the presence of normal bicarbonate/CO2. In the nominal absence of bicarbonate/CO2 fluid secretion was 18% of control. Vasopressin-induced fluid secretion was significantly inhibited by pretreatment of cysts with 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Cyst cell shrinkage in isosmotic chloride-free Ringer's solution (chloride replaced by gluconate) was inhibited by 0.1 mM basolateral DIDS. The results suggest that chloride-bicarbonate exchange in the basolateral membrane of MDCK cyst epithelial cells plays a critical role in cyst fluid secretion.

scholarly journals A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

2009 ◽  
Vol 184 (5) ◽  
pp. 721-736 ◽  
Author(s):  
Ruth Rollason ◽  
Viktor Korolchuk ◽  
Clare Hamilton ◽  
Mark Jepson ◽  
George Banting
Keyword(s):  
Epithelial Cells ◽  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Basolateral Membrane ◽  
Critical Role ◽  
Integral Membrane Protein ◽  
Apical Surface ◽  
Actin Network ◽  
Cell Height ◽  
Polarized Epithelial Cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.

scholarly journals The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

1998 ◽  
Vol 142 (5) ◽  
pp. 1245-1256 ◽  
Author(s):  
Jen-Zen Chuang ◽  
Ching-Hwa Sung
Keyword(s):  
Secretory Pathway ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Apical Plasma Membrane ◽  
Glutathione S Transferase ◽  
Sorting Signals ◽  
Apical Sorting

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

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