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• W2039156965 abstract "A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase β subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the β subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase β subunits associate with the endogenous Na,K-ATPase α1 subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase β subunit with the Na,K-ATPase α1 subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the β subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase β subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the β subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric β subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase β subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis. A dynamic equilibrium between multiple sorting pathways maintains polarized distribution of plasma membrane proteins in epithelia. To identify sorting pathways for plasma membrane delivery of the gastric H,K-ATPase β subunit in polarized cells, the protein was expressed as a yellow fluorescent protein N-terminal construct in Madin-Darby canine kidney (MDCK) and LLC-PK1 cells. Confocal microscopy and surface-selective biotinylation showed that 80% of the surface amount of the β subunit was present on the apical membrane in LLC-PK1 cells, but only 40% was present in MDCK cells. Nondenaturing gel electrophoresis of the isolated membranes showed that a significant fraction of the H,K-ATPase β subunits associate with the endogenous Na,K-ATPase α1 subunits in MDCK but not in LLC-PK cells. Hence, co-sorting of the H,K-ATPase β subunit with the Na,K-ATPase α1 subunit to the basolateral membrane in MDCK cells may determine the differential distribution of the β subunit in these two cell types. The major fraction of unassociated monomeric H,K-ATPase β subunits is detected in the apical membrane. Quantitative analysis showed that half of the apical pool of the β subunit originates directly from the trans-Golgi network and the other half from transcytosis via the basolateral membrane in MDCK cells. A minor fraction of monomeric β subunits detected in the basolateral membrane represents a transient pool of the protein that undergoes transcytosis to the apical membrane. Hence, the steady state distribution of the H,K-ATPase β subunit in polarized cells depends on the balance between (a) direct sorting from the trans-Golgi network, (b) secondary associative sorting with a partner protein, and (c) transcytosis. Integral membrane proteins located on the apical and basolateral membranes of epithelial cells reach their destination by one of two pathways. They can be sorted within the trans-Golgi network (TGN) 1The abbreviations used are: TGN, trans-Golgi network; ER, endoplasmic reticulum; CHX, cycloheximide; YFP-β, the fusion protein between the yellow fluorescent protein and the H,K-ATPase β subunit; NaKα, the Na,K-ATPase α1 subunit; NaKβ, the Na,K-ATPase β subunit; PNGF, peptide N-glycosidase F; MDCK, Madin-Darby canine kidney; PIPES, 1,4-piperazinediethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.1The abbreviations used are: TGN, trans-Golgi network; ER, endoplasmic reticulum; CHX, cycloheximide; YFP-β, the fusion protein between the yellow fluorescent protein and the H,K-ATPase β subunit; NaKα, the Na,K-ATPase α1 subunit; NaKβ, the Na,K-ATPase β subunit; PNGF, peptide N-glycosidase F; MDCK, Madin-Darby canine kidney; PIPES, 1,4-piperazinediethanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. into specific containers, which are then delivered directly either to the apical or basolateral membranes (1Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (151) Google Scholar, 2Nelson W.J. Yeaman C. Trends Cell Biol. 2001; 11: 483-486Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Mostov K. Su T. ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (252) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar, 5Rodriguez-Boulan E. Musch A. Le Bivic A. Curr. Opin. Cell Biol. 2004; 16: 436-442Crossref PubMed Scopus (45) Google Scholar). Alternatively, they are initially delivered first to one membrane (e.g. the basolateral membrane) but then retrieved from that membrane by the process of endocytosis and then delivered to the opposite membrane (i.e. the apical membrane). This latter indirect process has been termed transcytosis (3Mostov K. Su T. ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (252) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar, 5Rodriguez-Boulan E. Musch A. Le Bivic A. Curr. Opin. Cell Biol. 2004; 16: 436-442Crossref PubMed Scopus (45) Google Scholar).Both of these direct and indirect pathways are regulated. Specific machinery essential for the sorting process is present within the cell and recognizes sorting signals such as minimal amino acid motifs, carbohydrate or lipid moieties, embedded within individual proteins (1Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (151) Google Scholar, 2Nelson W.J. Yeaman C. Trends Cell Biol. 2001; 11: 483-486Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Mostov K. Su T. ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (252) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar, 5Rodriguez-Boulan E. Musch A. Le Bivic A. Curr. Opin. Cell Biol. 2004; 16: 436-442Crossref PubMed Scopus (45) Google Scholar). Apical sorting signals are usually found in the ectodomain or the transmembrane domain of proteins and may include glycosylphosphatidylinositol anchors, N- and O-linked glycans, and transmembrane anchor signals (1Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (151) Google Scholar, 2Nelson W.J. Yeaman C. Trends Cell Biol. 2001; 11: 483-486Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Mostov K. Su T. ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (252) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar, 5Rodriguez-Boulan E. Musch A. Le Bivic A. Curr. Opin. Cell Biol. 2004; 16: 436-442Crossref PubMed Scopus (45) Google Scholar). Basolateral sorting signals are usually found within the juxtamembrane cytoplasmic region of membrane proteins and include tyrosine-based and dileucine-based motifs (1Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (151) Google Scholar, 2Nelson W.J. Yeaman C. Trends Cell Biol. 2001; 11: 483-486Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 3Mostov K. Su T. ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (252) Google Scholar, 4Nelson W.J. Rodriguez-Boulan E. Nat. Cell Biol. 2004; 6: 282-284Crossref PubMed Scopus (16) Google Scholar, 5Rodriguez-Boulan E. Musch A. Le Bivic A. Curr. Opin. Cell Biol. 2004; 16: 436-442Crossref PubMed Scopus (45) Google Scholar). Several different sorting signals can be present in a protein, but they may vary in their importance in the targeting process. A particular motif might be preferentially recognized by the sorting machinery or be dominant over other signals (1Ikonen E. Simons K. Semin. Cell Dev. Biol. 1998; 9: 503-509Crossref PubMed Scopus (151) Google Scholar). Targeting of heterodimeric proteins may depend on signals embedded in one of the subunits, with the other subunit tagging along after synthesis of stable complexes in the ER (6Muth T.R. Gottardi C.J. Roush D.L. Caplan M.J. Am. J. Physiol. 1998; 274: C688-C696Crossref PubMed Google Scholar).The gastric H,K-ATPase and the Na,K-ATPase are two homologous transport enzymes that go through a cycle of phosphorylation and dephosphorylation coupled to ion transport, protons or hydronium in exchange for potassium (H,K-ATPase) and sodium in exchange for potassium (Na,K-ATPase). Both enzymes are heterodimers consisting of an α subunit, which contains the catalytic site, and a glycosylated β subunit, which is thought to be necessary for normal maturation and delivery of the enzyme out of the ER (7Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (265) Google Scholar). The Na,K-ATPase α subunit was shown to form a stable complex also with the H,K-ATPase β subunit that, similar to its natural partner, enabled maturation and plasma membrane delivery of the Na,K-ATPase α subunit in various cell expression systems (8Koenderink J.B. Swarts H.G. Hermsen H.P. De Pont J.J. J. Biol. Chem. 1999; 274: 11604-11610Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 9Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10Eakle K.A. Kim K.S. Kabalin M.A. Farley R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2834-2838Crossref PubMed Scopus (75) Google Scholar, 11Geering K. Crambert G. Yu C. Korneenko T.V. Pestov N.B. Modyanov N.N. Biochemistry. 2000; 39: 12688-12698Crossref PubMed Scopus (27) Google Scholar, 12Horisberger J.D. Jaunin P. Reuben M.A. Lasater L.S. Chow D.C. Forte J.G. Sachs G. Rossier B.C. Geering K. J. Biol. Chem. 1991; 266: 19131-19134Abstract Full Text PDF PubMed Google Scholar, 13Noguchi S. Maeda M. Futai M. Kawamura M. Biochem. Biophys. Res. Commun. 1992; 182: 659-666Crossref PubMed Scopus (25) Google Scholar). Despite the similarities in structure and catalytic properties, the Na,K-ATPase and H,K-ATPase reside in the opposite membrane domains in epithelial cells. The Na,K-ATPase is targeted to basolateral surfaces in most polarized cells (14Caplan M.J. Am. J. Physiol. 1997; 272: G1304-G1313PubMed Google Scholar). In contrast, the gastric H,K-ATPase, the enzyme responsible for acid secretion by the stomach, is located in tubulovesicular elements in the resting parietal cell and relocates to the secretory canalicular (apical) membrane upon stimulation of acid secretion (15Smolka A. Helander H.F. Sachs G. Am. J. Physiol. 1983; 245: G589-G596PubMed Google Scholar, 16Urushidani T. Forte J.G. Am. J. Physiol. 1987; 252: G458-G465PubMed Google Scholar). The nature of the targeting information contained within each subunit has been deduced from studies of MDCK and LLC-PKI cells in which both subunits are coexpressed or the β subunit is expressed alone. Studies in which the α subunit is expressed alone cannot be done, because this subunit is degraded and fails to be delivered to the plasma membrane (7Geering K. J. Bioenerg. Biomembr. 2001; 33: 425-438Crossref PubMed Scopus (265) Google Scholar).Coexpression studies in LLC-PK1 cells demonstrated the presence of apical sorting signals in both the α and β subunits (17Gottardi C.J. Caplan M.J. J. Cell Biol. 1993; 121: 283-293Crossref PubMed Scopus (120) Google Scholar) of the H,K-ATPase and a basolateral sorting signal in the α1 subunit of the Na,K-ATPase (6Muth T.R. Gottardi C.J. Roush D.L. Caplan M.J. Am. J. Physiol. 1998; 274: C688-C696Crossref PubMed Google Scholar). Sorting signals in both Na,K-ATPase and H,K-ATPase catalytic subunits were shown to reside in their N-terminal halves (6Muth T.R. Gottardi C.J. Roush D.L. Caplan M.J. Am. J. Physiol. 1998; 274: C688-C696Crossref PubMed Google Scholar, 17Gottardi C.J. Caplan M.J. J. Cell Biol. 1993; 121: 283-293Crossref PubMed Scopus (120) Google Scholar). An apical sorting signal of the H,K-ATPase β subunit was demonstrated to be encoded in its N-glycosylation sites (18Vagin O. Turdikulova S. Sachs G. J. Biol. Chem. 2004; 279: 39026-39034Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). On the other hand, when the H,K-ATPase β subunit is expressed alone, it can be distributed to either the apical or the basolateral membrane, depending on the cell type examined. It is distributed to the apical membrane in LLC-PK1 cells and mostly to the basolateral membrane in MDCK cells (19Roush D.L. Gottardi C.J. Naim H.Y. Roth M.G. Caplan M.J. J. Biol. Chem. 1998; 273: 26862-26869Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These results indicate the β subunit may contain both apical and basolateral sorting signals.Therefore, the gastric H,K-ATPase β subunit has several properties that make it an excellent model to use in studies of the sorting pathways involved in distribution of membrane proteins and the factors modulating the sorting process. It can be expressed alone or in association with its natural partner or other subunits in cultured cells, it has embedded in it both apical and basolateral sorting signals, and it is delivered predominantly to the apical or basolateral membranes in different polarized cell systems.In the present studies, the H,K-ATPase β subunit was expressed as a YFP N-terminal fusion protein in MDCK and LLC-PK1 cells. Using confocal microscopy, surface-selective biotinylation, and biotin cleavage, we were able to assess the steady state distribution of the protein, determine its fate after it was inserted into either membrane domain, and measure accumulation of the newly delivered protein in the apical membrane. The results indicate the presence of both direct and indirect routes for apical delivery of the protein. By selectively inhibiting a direct or indirect apical pathway, we were able to evaluate the relative quantity of the β subunit, which was directly delivered from the TGN to the apical membrane compared with that which arrived there by transcytosis from the basolateral membrane. Using nondenaturing gel electrophoresis, we determined whether association of the expressed protein with the endogenous partner affects its sorting and final distribution. We found that promiscuous association with the endogenous Na,K-ATPase α1 subunit in MDCK but not LLC-PK1 cells is probably responsible for differential distribution of the H,K-ATPase β subunit in the two cell types.The results of these studies indicate that membrane proteins containing both apical and basolateral sorting signals, such as the gastric H,K-ATPase β subunit, which are resident in the apical membrane, can arrive there by both direct delivery from the TGN and transcytosis via the basolateral membrane. Moreover, association with another subunit, if this has a dominant sorting signal, can cause it to be delivered to a membrane different than in its native state. Furthermore, these data emphasize that sorting is a complex process involving an interaction between sorting machinery unique to specific cells, signals embedded in proteins, and, in the case of heterodimeric proteins, the relative dominance of the sorting information in the particular subunit.EXPERIMENTAL PROCEDURESConstruction of cDNAs Encoding YFP-β Fusion Proteins and Mutants Lacking Glycosylation Sites—pcDNA3(+)β (20Lambrecht N. Munson K. Vagin O. Sachs G. J. Biol. Chem. 2000; 275: 4041-4048Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) was used as a source for cDNA encoding the rabbit H,K-ATPase β-subunit (21Reuben M.A. Lasater L.S. Sachs G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6767-6771Crossref PubMed Scopus (136) Google Scholar) (GenBank™ accession number M35544). The cDNA encoding the β-subunit was inserted into the multiple cloning site of the expression vector pEYFP-C1 (BD Bioscience Clontech) using BglII and BamHI restriction sites to form pEYFP-β that encodes YFP-β, a fusion protein of YFP linked to the amino terminus of the H,K-ATPase β-subunit. Mutants were generated by using the QuikChange mutagenesis kit (Stratagene), using pEYFP-β as a template.Stable Transfection—In order to obtain cell lines stably expressing wild type YFP-β or mutant YFP-β fusion proteins, LLC-PK1 cells were grown on 10-cm plates until 20% confluent and transfected with wild type or mutant pEYFP-β using FuGENE 6 Transfection Reagent (Roche Applied Science). Stable cell lines were selected by adding, 24 h after transfection, the eukaryotic selection marker G-418 at a final concentration of 1.0 mg/ml. This concentration of G-418 was maintained until single colonies appeared. 15–20 colonies were isolated, expanded, and grown in the presence of 0.25 mg of G-418/ml of medium in a 24-well plate. Two clones with the highest expression of YFP-β were selected and expanded for further studies.The cell lines expressing YFP-β were subjected to a second transfection with pcDNA3.1 (zeo+)α encoding the rabbit H,K-ATPase α subunit (GenBank™ accession number X64694). By addition of the second selection marker zeocin at a concentration of 0.4 mg/ml in addition to maintenance of a concentration of G-418 of 0.25 mg/ml, 15–20 cell lines expressing both α- and β-subunits were selected. Two clones with the best ratio of expressed H,K-ATPase to total protein were expanded for biotinylation experiments. The maintenance concentration for zeocin was 0.1 mg/ml.Confocal Microscopy Identification of Site of Expression of YFP-β— Cells stably expressing wild type or mutant YFP-β were grown for at least 5 days after becoming confluent on glass bottom microwell dishes (MatTek Corp.). Confocal microscopic images were acquired using the Zeiss LSM 510 laser-scanning confocal microscope using LSM 510 software, version 3.2.Estimation of Surface YFP-β Content by Surface-specific Biotinylation—LLC-PK1 cells stably expressing wild type or mutant YFP-β were maintained for at least 5 days after becoming confluent in Corning Costar brand polyester transwell inserts (Corning Glass) in 6-well plates. Biotinylation of the apical or basolateral membrane proteins was performed by previously described procedures (22Gottardi C.J. Dunbar L.A. Caplan M.J. Am. J. Physiol. 1995; 268: F285-F295Crossref PubMed Google Scholar, 23Kroepfl J.F. Gardinier M.V. J. Neurochem. 2001; 77: 1301-1309Crossref PubMed Scopus (22) Google Scholar). Briefly, cell monolayers were biotinylated with EZ-Link™ sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (Pierce) that was added from either the apical or basolateral side. After quenching the biotinylation reaction, cells were washed and then lysed, and membranes were solubilized by incubation with 200 μl of 0.15 m NaCl in 15 mm Tris, pH 8.0, with 1% Triton X-100 and 4 mm EGTA. Cell lysates were clarified by centrifugation (15,000 × g, 10 min). Samples containing 20 μl of supernatant mixed with 15 μl of SDS-containing sample buffer were loaded onto SDS-polyacrylamide gel to determine the total YFP-β content in the supernatant. To isolate biotinylated proteins, the rest of each supernatant was incubated with 100 μl of streptavidin-agarose beads (Sigma) in a total volume of 800 μl of the lysing buffer for 1 h at 4 °C with continuous rotation. The bead-adherent complexes were washed three times on the beads, and then proteins were eluted from the beads by incubation in 40 μl of SDS-PAGE sample buffer (4% SDS, 0.05% bromphenol blue, 20% glycerol, 1% β-mercaptoethanol in 0.1 m Tris, pH 6.8) for 5 min at 80 °C, separated on SDS-polyacrylamide gel, and analyzed by Western blot using 2B6 monoclonal antibody against the H,K-ATPase β subunit (MBL, Inc.) or the monoclonal antibody against the Na,K-ATPase β1 subunit (Novus Biologicals), the monoclonal antibody against the Na,K-ATPase α1 subunit (Upstate Biotechnology, Inc., Lake Placid, NY), or the monoclonal antibody against the H,K-ATPase subunit (monoclonal antibody 12.18; a generous gift from Dr. A. Smolka) as a primary antibody and anti-mouse IgG conjugated to alkaline phosphatase (Promega) as the secondary antibody according to the manufacturer's instructions. Immunoblots were quantified by densitometry using Kodak 1D 3.6 software.In all experiments, the specific basolateral location of the Na,K-ATPase β1 subunit was considered as a control for intact tight junctions, and the absence of high mannose type YFP-β in biotinylated samples was used as an indication of plasma membrane integrity during biotinylation. The presence of biotinylated high mannose type YFP-β protein would show that the biotinylation reagent had access to the intracellular pool of high mannose type YFP-β because of plasma membrane leak to the reagent during the experiment (18Vagin O. Turdikulova S. Sachs G. J. Biol. Chem. 2004; 279: 39026-39034Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Endocytosis and Recycling Assay by Surface-selective Biotinylation and Surface-selective Biotin Cleavage—Polarized cells stably expressing wild type YFP-β were biotinylated from either apical or basolateral side as described above with different incubation times. Cells were then incubated at 18 °C to impede further apical membrane delivery of intracellular protein for 20, 60, or 120 min. After this low temperature incubation, apical biotin was stripped off (Fig. 4A) by incubating with 50 mm reduced glutathione (Sigma) in 100 mm NaCl with 10% fetal bovine serum, pH 8.4, twice for 20 min. This procedure selected for internalized biotinylated protein. After cell lysis, the previously internalized biotinylated proteins were precipitated, washed, eluted from streptavidinagarose beads, and analyzed by SDS-polyacrylamide gel and Western blot analysis as described above. In the negative control, biotin was stripped off immediately after biotinylation. To determine the total biotinylated YFP-β, in the positive control, cells were lysed immediately after biotinylation. To account for any instability of biotinylated protein, in a separate experiment, cells were incubated at 18 °C for 120 min and then lysed.To measure recycling, cells were incubated at 18 °C for 120 min, and the surface biotin was removed as above. Then these cells that now only contained internalized YFP-β were incubated at 37 °C for 30 min (Fig. 4A) to estimate delivery of this fraction to the plasma membrane. After this procedure, in the control, cells were lysed immediately, whereas in the experimental cells, surface biotin was stripped off again to account for the fraction of internalized YFP-β that was returned to the plasma membrane. After cell lysis, biotinylated proteins were precipitated, washed, eluted, and analyzed as described above.Transcytosis Assay by Surface-selective Biotinylation and Surface-selective Biotin Cleavage—A diagram of the experimental procedure to measure transcytosis from the apical to the basolateral membrane is shown in Fig. 5A. Filter-grown MDCK cells expressing the wild type YFP-β were biotinylated from either apical or basolateral side. After a 4-h incubation at 37 °C in the control experiment, cells were lysed immediately. In the three separate experiments, biotin was stripped off by incubating with 50 mm reduced glutathione from the apical surface only, from the basolateral surface only, or from both the apical and basolateral surfaces before cell lysis. Biotinylated proteins were precipitated and analyzed as described above. The amount of protein that was transcytosed to the opposite membrane domain was calculated as the difference between the amount detected in the experiment where biotin was stripped off from the side of biotinylation and the amount detected in the experiment where biotin was stripped off from both sides. Transcytosis from the basolateral to the apical membrane was measured according to a similar scheme, with the only difference being that the cells were biotinylated from the basolateral surface rather than the apical surface.Fig. 5Detection of transcytosis from the apical to the basolateral membrane in MDCK cells. MDCK cells expressing the wild type YFP-β were grown on transwell inserts. A, scheme showing the experimental procedure. Cells were biotinylated from the apical side. T0, total biotinylated YFP-β (positive control); N, biotin was stripped off immediately after biotinylation (negative control); T, cells were incubated at 37 °C for 4 h (control for stability of biotinylated YFP-β); in the next three inserts, after cell incubation at 37 °C for 4 h, biotin was stripped off from the apical side only (T–A), from both apical and basolateral sides (I), or from the basolateral side only (T–B). After completion of the above procedures and cell lysis, biotinylated proteins were precipitated on streptavidin-agarose beads and analyzed by Western blot. B, a representative immunoblot and quantification of the data from three independent experiments. C, calculation of the amounts of YFP-β that were transcytosed to the basolateral membrane, internalized, or retained in the apical membrane after a 4-h incubation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Blue Native Gel of Microsomal Membranes Isolated from MDCK and LLC-PK1 Cells to Detect Associated Na,K-ATPase—The microsomal membranes from MDCK and LLC-PK1 cells stably expressing YFP-β were isolated as described before (24Vagin O. Denevich S. Sachs G. Am. J. Physiol. 2003; 285: C968-C976Crossref Scopus (23) Google Scholar). Briefly, cells were collected in buffer A (10 mm PIPES/Tris, pH 7.0, with 2 mm EGTA and 2 mm EDTA) containing 250 mm sucrose, homogenized, layered onto the 42% sucrose solution, and spun in the Beckman SW28 swinging bucket rotor at 25,000 rpm for 1 h at 4 °C. The fraction at the buffer/sucrose interface was collected, diluted with buffer A, and centrifuged in a Beckman 75Ti rotor (35,000 rpm, 4 °C, 1 h). The pellet was resuspended in 2 ml of buffer A and homogenized with a Teflon homogenizer (Wheaton, Millwille, NY). The total protein concentration was determined using a modified Lowry protein assay reagent (Pierce). The typical protein concentration was 5–10 μg/μl.Blue native gel electrophoresis was performed as previously described (25Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1885) Google Scholar). All buffers and solutions were at pH 7.0 at 4 °C. 1 mg of protein of microsomal membranes isolated from MDCK or LLC-PK1 cells was resuspended in 100 μl of 50 mm BisTris buffer containing 0.75 m aminocaproic acid (Fluka). After adding 12.5 μl of 10% n-dodecyl-β-d-maltoside (Roche Applied Science) and a 20-min incubation on ice with vortexing every 5 min, samples were centrifuged at 14,000 × g for 10 min. Then 6.3 μl of a 5% suspension of Coomassie Brilliant Blue G-250 (Serva, Germany) in 0.5 m aminocaproic acid was added to 100 μl of supernatant. Samples were then stored on ice for no more than 30 min prior to gel loading. A 4–12% gradient gel with 4% stacker was used (Invitrogen). The anode buffer contained 50 mm BisTris. The cathode buffer contained 50 mm Tricine, 15 mm BisTris, and 0.01% Coomassie Brilliant Blue G-250. The gel, buffers, and electrophoretic apparatus were chilled to 4 °C before samples were loaded (35 μl/well with 40–80 μg of protein). Electrophoresis was carried out at 34 V and 0.05 mA overnight. Then the cathode buffer was exchanged for one-tenth the amount of 0.001% Coomassie Blue, and electrophoresis was resumed at 60 V, 0.05 mA. Staining was carried out as described above for the various ATPase subunits.RESULTSSteady State Surface Distribution of the Wild Type and Mutant YFP-β in Polarized MDCK and LLC-PK1 Cells—Expression of the gastric H,K-ATPase β subunit as a YFP N-terminal construct in eukaryotic cells results in a synthesis of a fusion protein that retains correct folding, post-translational modifications, and ability to assemble with its α subunit and supports the active conformation of the α,β complex, as was shown before in nonpolarized HEK-293 cells (24Vagin O. Denevich S. Sachs G. Am. J. Physiol. 2003; 285: C968-C976Crossref Scopus (23) Google Scholar) and polarized LLC-PK1 cells (18Vagin O. Turdikulova S. Sachs G. J. Biol. Chem. 2004; 279: 39026-39034Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The expressed fusion protein of YFP and the H,K-ATPase β subunit (YFP-β) was detected by Western blot in cell lysates of MDCK cells as two bands, a major band at 80–100 kDa and a minor band at ∼75 kDa (Fig. 1A, MDCK, wt, lane T) similar to the pattern found previously in HEK-293 and LLC-PK1 cells (18Vagin O. Turdikulova S. Sachs G. J. Biol. Chem. 2004; 279: 39026-39034Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Vagin O. Denevich S. Sachs G. Am. J. Physiol. 2003; 285: C968-C976Crossref Scopus (23) Google Scholar). A similar pattern was observed in the cell lines expressing the wild type protein and Y20A and Y20F mutants (Fig. 1A, lanes T). The lower H YFP-β band is endoglycosidas" @default.
• W2039156965 created "2016-06-24" @default.
• W2039156965 creator A5011647157 @default.
• W2039156965 creator A5019676161 @default.
• W2039156965 creator A5057185561 @default.
• W2039156965 creator A5082253629 @default.
• W2039156965 date "2005-04-01" @default.
• W2039156965 modified "2023-10-15" @default.
• W2039156965 title "Use of the H,K-ATPase β Subunit to Identify Multiple Sorting Pathways for Plasma Membrane Delivery in Polarized Cells" @default.
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