Artigo Acesso aberto Revisado por pares

Rab13 Mediates the Continuous Endocytic Recycling of Occludin to the Cell Surface

2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês

10.1074/jbc.m406906200

ISSN

1083-351X

Autores

Shinya Morimoto, Noriyuki Nishimura, Tomoya Terai, S Manabe, Yasuyo Yamamoto, Wakako Shinahara, Hidenori Miyake, Seiki Tashiro, Mitsuo Shimada, Takuya Sasaki,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells. During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells. Polarized epithelial cells create apical and basolateral plasma membrane (PM) 1The abbreviations used are: PM, plasma membrane; Ad, adenovirus; AJ, adherens junction; BAF, bafilomycin A1; BHK, baby hamster kidney; CHX, cycloheximide; EGFP, enhanced green fluorescence protein; EMT, epithelial-mesenchymal transitions; GFP, green fluorescence protein; HA, hemagglutinin; MDCK, Madin-Darby canine kidney; MESNA, 2-mercaptoethanesulfonic acid; MET, mesenchymal-epithelial transitions; PBS, phosphate-buffered saline; pIgR, polymeric immunoglobulin receptor; TfR, transferrin receptor; TJ, tight junction; APMSF, p-amidinophenyl methanesulfonyl fluoride. domains that face the lumen and basement membrane, respectively. These two domains have distinct functional properties and display unique lipid and protein compositions. Tight junctions (TJs) act as a fence preventing the lateral diffusion of proteins and lipids between the two domains and as a gate regulating solute flux through the paracellular space (1Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2041) Google Scholar). Proteins constituting TJs include the transmembrane proteins mediating cell-cell adhesion and the cytosolic plaque proteins linking TJs to the cytoskeleton and participating in intracellular signaling (2Gonzalez-Mariscal L. Betanzos A. Nava P. Jaramillo B.E. Prog. Biophys. Mol. Biol. 2003; 81: 1-44Crossref PubMed Scopus (921) Google Scholar). Occludin, claudins, and junctional adhesion molecules constitute the transmembrane proteins in TJs, whereas a number of scaffolding proteins and signaling molecules such as zonula occludens-1 and Par3-Par6-atypical protein kinase C complex have been identified as the cytosolic plaque proteins in TJs (3Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1285) Google Scholar, 4Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2128) Google Scholar, 5Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1714) Google Scholar, 6Izumi Y. Hirose T. Tamai Y. Hirai S. Nagashima Y. Fujimoto T. Tabuse Y. Kemphues K.J. Ohno S. J. Cell Biol. 1998; 143: 95-106Crossref PubMed Scopus (442) Google Scholar, 7Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Ruco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1147) Google Scholar, 8Suzuki A. Yamanaka T. Hirose T. Manabe N. Mizuno K. Shimizu M. Akimoto K. Izumi Y. Ohnishi T. Ohno S. J. Cell Biol. 2001; 152: 1183-1196Crossref PubMed Scopus (383) Google Scholar). However, the molecular mechanisms governing the de novo formation of TJs are poorly understood. The maintenance of epithelial polarity depends on the continuous sorting of membrane proteins and lipids into distinct cell surface domains. This sorting occurs both in the trans-Golgi network during biosynthetic vesicular transport and, after endocytosis, in endosomes (9Mostov K. Su T. Ter Beest M. Nat. Cell Biol. 2003; 5: 287-293Crossref PubMed Scopus (255) Google Scholar, 10Zegers M.M. O'Brien L.E. Yu W. Datta A. Mostov K.E. Trends Cell Biol. 2003; 13: 169-176Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). These polarized vesicular transport pathways must be strictly regulated to ensure the controlled loss and recovery of epithelial polarity during dynamic morphogenetic events. The Rab family small G proteins consist of more than 60 family members in mammalian cells and play a crucial role in determining the specificity of vesicular transport pathways. Each Rab family member that is localized to a distinct compartment in the exocytic or endocytic pathway functions as a molecular switch, cycling between the GTP- and GDP-bound conformations, at the compartment where they reside (11Pfeffer S.R. Trends Cell Biol. 2001; 11: 487-491Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 12Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2061) Google Scholar, 13Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2710) Google Scholar). Two Rab family members, Rab3B and Rab13, localize to TJs, and it has been proposed that they function as putative regulators of polarized vesicular transport to and/or from TJs in polarized epithelial cells. Indeed, recent evidence suggests that Rab13 regulates the assembly of functional TJs and that the role of Rab13 in polarized vesicular transport from the trans-Golgi network to the cell surface is distinct from that of Rab3B (14Weber E. Berta G. Tousson A. John P. Green M.W. Gopalokrishnan U. Jilling T. Sorscher E.J. Elton T.S. Abrahamson D.R. Kirk K.L. J. Cell Biol. 1994; 125: 583-594Crossref PubMed Scopus (169) Google Scholar, 15Zahraoui A. Joberty G. Arpin M. Fontaine J.J. Hellio R. Tavitian A. Louvard D. J. Cell Biol. 1994; 124: 101-115Crossref PubMed Scopus (203) Google Scholar, 16Marzesco A.M. Dunia I. Pandjaitan R. Recouvreur M. Dauzonne D. Benedetti E.L. Louvard D. Zahraoui A. Mol. Biol. Cell. 2002; 13: 1819-1831Crossref PubMed Scopus (98) Google Scholar, 17Yamamoto Y. Nishimura N. Morimoto S. Kitamura H. Manabe S. Kanayama H. Kagawa S. Sasaki T. Biochem. Biophys. Res. Commun. 2003; 308: 270-275Crossref PubMed Scopus (40) Google Scholar). Adherens junctions (AJs) and TJs are points of adhesion between epithelial cells that ensure the maintenance of appropriate epithelial cell polarity. Cadherins are essential adhesion molecules within AJs supporting not only stable cell-cell contacts but also dynamic morphogenetic events such as epithelial-mesenchymal transitions (EMT) and mesenchymal-epithelial transitions (MET) (18Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1258) Google Scholar). During EMT, E-cadherin, the prototypical epithelial cadherin, is down-regulated by transcriptional silencing and/or protein degradation through the ubiquitin-proteasome pathway (19Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar). The endocytosis and recycling of E-cadherin have recently emerged as alternative mechanisms allowing cells to undergo rapid changes in morphology in response to extracellular stimuli (20Kamei T. Matozaki T. Sakisaka T. Kodama A. Yokoyama S. Peng Y.F. Nakano K. Takaishi K. Takai Y. Oncogene. 1999; 18: 6776-6784Crossref PubMed Scopus (176) Google Scholar, 21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar, 22Paterson A.D. Parton R.G. Ferguson C. Stow J.L. Yap A.S. J. Biol. Chem. 2003; 278: 21050-21057Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The small G protein ARF6 is implicated in the regulation of the endocytic recycling of E-cadherin (23Palacios F. Price L. Schweitzer J. Collard J.G. D'Souza-Schorey C. EMBO J. 2001; 20: 4973-4986Crossref PubMed Scopus (250) Google Scholar). Among the transmembrane proteins in TJs, occludin and claudins, a family composed of more than 20 different members, are thought to be responsible for the formation of TJs and epithelial polarization (4Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2128) Google Scholar, 5Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1714) Google Scholar). Like cadherins, it has been shown that both occludin and claudins are transcriptionally silenced during EMT, and occludin is subject to post-transcriptional protein degradation by the ubiquitin-proteasome pathway (24Traweger A. Fang D. Liu Y.C. Stelzhammer W. Krizbai I.A. Fresser F. Bauer H.C. Bauer H. J. Biol. Chem. 2002; 277: 10201-10208Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 25Ikenouchi J. Matsuda M. Furuse M. Tsukita S. J. Cell Sci. 2003; 116: 1959-1967Crossref PubMed Scopus (541) Google Scholar). To begin to understand the molecular mechanisms underlying the dynamic change in epithelial polarity during EMT and/or MET, we examined the intracellular transport of occludin and claudins to and from the cell surface in both fibroblastic BHK and epithelial MTD-1A cells. When occludin was expressed in BHK cells, it was continuously endocytosed and recycled back to the cell surface. Endogenous occludin was also subjected to continuous endocytic recycling in MTD-1A cells. Our results indicate that Rab13, one of the cytosolic plaque proteins in TJs, directs the continuous endocytic recycling of occludin in both fibroblastic and epithelial cells. Cell Culture and Transfection—MTD-1A cells were kindly supplied by Dr. S. Tsukita (Kyoto University, Kyoto, Japan). BHK, HeLa, MDCK, and Caco2 cells were obtained from ATCC (Manassas, VA). MTD-1A and BHK cells were cultured at 37 °C (5% CO2 and 95% air) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. MTD-1A cells were transfected using FuGENE 6 transfection reagent (Roche Applied Science). Antibodies—Rat anti-occludin (MOC37) and rabbit anti-polymeric immunoglobulin receptor (pIgR) antibodies were kind gifts from Dr. S. Tsukita (Kyoto University, Kyoto, Japan) and Dr. A. L. Hubbard (Johns Hopkins University, Baltimore, MD). Rabbit anti-claudin-1, rabbit anti-occludin, rabbit anti-Rab11, and mouse anti-transferrin receptor (TfR) antibodies were purchased from Zymed Laboratories Inc. (San Francisco). Rabbit anti-GFP and mouse anti-GFP were from Molecular Probes (Eugene, OR). Mouse anti-HA (12CA5) and rat anti-HA (3F10) were from Roche Applied Science. Mouse anti-Rab4 was from BD Transduction Laboratories (Lexington, KY), and mouse anti-FLAG (M2) was from Sigma. Construction of Expression Plasmids—Bovine Rab3B cDNA was a kind gift from Dr. Y. Takai (Osaka University, Osaka, Japan). Rab4, Rab11, Rab13, TfR, claudin-1, and occludin cDNAs were isolated by reverse transcription-PCR from MDCK cells, rat brain, Caco2 cells, HeLa cells, mouse kidney, and mouse liver, respectively. Dominant active and negative mutants of Rab3B (Rab3B Q81L and Rab3B T36N) and Rab13 (Rab13 Q67L and Rab13 T22N) were constructed using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA). All cDNAs used in this study were cloned into the N-terminal HA-tagged (pCI-neo-HA), the N-terminal EGFP-tagged (pCR259-EGFP), the C-terminal FLAG-tagged (pBS-FLAG), or pCR3.1 expression plasmid and sequenced using an automated DNA sequencer 377 (Applied Biosystems). Recombinant T7 Vaccinia Virus System—Recombinant vaccinia virus-expressing T7 RNA polymerase (vTF7-3) was described previously (26Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (398) Google Scholar). BHK cells were infected with vTF7-3 at a multiplicity of infection of 10 for 30 min and then transfected with either pCR3.1-TfR, pBS-claudin-1-FLAG, or pBS-occludin-FLAG alone or in combination with pCI-neo-HA (mock), pCI-neo-HA-Rab3B T36N, pCI-neo-HA-Rab3B Q81L, pCI-neo-HA-Rab13 T22N, or pCI-neo-HA-Rab13 Q67L. After a 6-h transfection, cells were subjected either to endocytosis or recycling assay. Recombinant Adenovirus Infection—Recombinant adenoviruses expressing EGFP and EGFP-Rab13 Q67L (Ad-EGFP and Ad-EGFP-Rab13 Q67L) were constructed using the Transpose-Ad Adenoviral Vector System (Qbiogene, Carlsbad, CA) according to the manufacturer's instructions. Briefly, EGFP and EGFP-Rab13 Q67L cDNAs were cloned into a pCR259 transfer vector. Recombinant adenoviral plasmid was generated by Tn7-mediated transposition in Escherichia coli. The resulting plasmid was linearized by PacI digestion and transfected into QBI-HEK293 cells using an MBS Mammalian Transfection kit (Stratagene). After a 24-h transfection, cells were split into a 96-well plate and incubated at 37 °C for 10–14 days. Screening of recombinant adenovirus was done by PCR and immunoblot analysis. Recombinant adenovirus was amplified in QBI-HEK293 cells, and its titer was determined by multiplicity of infection test. MTD-1A cells were infected with Ad-EGFP (mock) or Ad-EGFP-Rab13 Q67L at a multiplicity of infection of 100. After a 36-h culture, cells were subjected either to endocytosis or recycling assay. Endocytosis Assay—Endocytosis assay was performed as described previously (21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar). Briefly, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-SS-Biotin (Pierce) in PBS containing 0.9 mm CaCl2 and 0.33 mm MgCl2 (PBS/CM) at 4 °C for 30 min, quenched with 50 mm NH4Cl in PBS/CM at 4 °C for 15 min, and incubated at 37 °C or 18 °C for the indicated periods of time to allow endocytosis. The remaining biotin on the cell surface was stripped with 50 mm MESNA in 100 mm Tris/HCl (pH 8.6) containing 100 mm NaCl and 2.5 mm CaCl2 at 4 °C for 30 min and quenched with 5 mg/ml iodoacetamide in PBS/CM at 4 °C for 15 min. After lysis with 50 mm Tris/HCl (pH 8.0) containing 1.25% Triton X-100, 0.25% SDS, 150 mm NaCl, 5 mm EDTA and 10 μg/ml APMSF, an aliquot was taken to determine the total amount of cargo proteins expressed in the cells. Biotinylated cargo proteins were then isolated with UltraLink Immobilized NeutrAvidin Plus beads (Pierce). The samples were prepared for immunoblot analysis. The values for biotinylated cargo proteins protected from MESNA treatment were normalized to total cargo proteins expressed in the cells. Immunoblot—Samples were separated on SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride membrane. Membrane blocking and antibody dilutions were done in Block Ace (Dainippon Pharmaceutical, Osaka, Japan). Blots were developed by chemiluminescence using horseradish peroxidase-coupled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and an ECL-Plus kit (Amersham Biosciences). Quantitation was performed after scanning of the autoradiograph film of nonsaturating signals by using a NIH Image 1.62 program. Potassium (K+) Depletion—Potassium depletion was performed as described previously (27Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar). Briefly, BHK cells were rinsed sequentially three times with K+-free buffer containing 20 mm Hepes/NaOH (pH 7.4), 140 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 1 mg/ml d-glucose, hypotonically shocked with hypotonic buffer containing 10 mm Hepes/NaOH (pH 7.4), 70 mm NaCl, 0.5 mm CaCl2, 0.5 mm MgCl2, and 0.5 mg/ml d-glucose, and incubated at 37 °C for 15 min with K+-free buffer to deplete K+. The endocytosis assay was performed using K+-free buffer instead of PBS/CM. Recycling Assay—Recycling assay was performed as described previously (21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar). Briefly, cell surface proteins were biotinylated with 0.5 mg/ml Sulfo-NHS-SS-Biotin (Pierce) in PBS/CM at 4 °C for 30 min, quenched with 50 mm NH4Cl in PBS/CM at 4 °C for 15 min, and incubated at 37 °C for the indicated periods of time to allow endocytosis. The remaining biotin on the cell surface was stripped with 50 mm MESNA in 100 mm Tris/HCl (pH 8.6) containing 100 mm NaCl and 2.5 mm CaCl2 at 4 °C for 30 min, and quenched with 5 mg/ml iodoacetamide in PBS/CM at 4 °C for 15 min. Cells were again incubated at 37 °C for the indicated periods of time to allow recycling of endocytosed cargo proteins back to the cell surface. Then newly appeared cell surface biotin was again stripped with 50 mm MESNA in 100 mm Tris/HCl (pH 8.6) containing 100 mm NaCl and 2.5 mm CaCl2 at 4 °C for 30 min, and quenched with 5 mg/ml iodoacetamide in PBS/CM at 4 °C for 15 min. After lysis with 50 mm Tris/HCl (pH 8.0) containing 1.25% Triton X-100, 0.25% SDS, 150 mm NaCl, 5 mm EDTA, and 10 μg/ml APMSF, an aliquot was taken to determine the total amount of cargo proteins expressed in the cells. Biotinylated cargo proteins were then isolated with UltraLink Immobilized NeutrAvidin Plus beads (Pierce). The samples were prepared for immunoblot analysis. The values for biotinylated cargo proteins protected from MESNA treatment were normalized to total cargo proteins expressed in the cells. Bafilomycin A1 (BAF) Treatment—BHK cells were preincubated in 1 μm BAF at 4 °C for 30 min and then incubated in 1 μm BAF at 37 °C for the indicated periods of time to allow recycling of endocytosed cargo proteins back to the cell surface. The recycling assay was performed as described above. Immunofluorescence Microscopy—MTD-1A cells grown on glass coverslips were fixed with either 1% formaldehyde in PBS for 15 min at room temperature (for immunostaining of occludin, occludin-HA, HA-GFP, HA-TfR, occludin-GFP, occludin-Rab4, occludin-Rab11, and occludin-TfR), -20 °C methanol for 5 min on ice (for immunostaining of claudin-1), or 10% trichloroacetic acid in PBS for 15 min on ice (for immunostaining of HA-claudin-1). After permeabilization with 0.2% Triton X-100 in PBS for 15 min and blocking with 5% goat serum in PBS for 60 min at room temperature, cells were incubated with primary antibodies for 60 min and with Alexa 488 or 594-conjugated secondary antibodies (Molecular Probes) for 60 min at room temperature. Fluorescent images of the cells were acquired using a Radiance 2000 confocal laser scanning microscope (Bio-Rad). Microinjection—MTD-1A cells were seeded onto glass coverslips and grown for 24 h. Either pCI-neo-HA-Rab3B T36N, pCI-neo-HA-Rab3B Q81L, pCI-neo-HA-Rab13 T22N, or pCI-neo-HA-Rab13 Q67L expression plasmid (0.05 mg/ml) was microinjected into the nuclei of the cells. Microinjections were performed using the Inject Man NI2 Micromanipulator and FemtoJet Microinjector Systems (Eppendorf, Hamburg, Germany) mounted on an inverted microscope TE-2000-U (Nikon, Tokyo, Japan). Nuclear injections were operated using a Z (depth) limit option, a 0.1-s injection time, and an injection pressure of 70 hectopascals. Cells were grown for an additional 18 h to allow the expression of proteins before fixation. Occludin Is Endocytosed Continuously in Fibroblastic BHK Cells—To begin to understand the molecular basis for the dynamic rearrangement of TJs, we first examined the transport of TJ membrane proteins to and from the cell surface in fibroblastic BHK cells (17Yamamoto Y. Nishimura N. Morimoto S. Kitamura H. Manabe S. Kanayama H. Kagawa S. Sasaki T. Biochem. Biophys. Res. Commun. 2003; 308: 270-275Crossref PubMed Scopus (40) Google Scholar). Although the transport of claudin-1, the original member of claudins, to the cell surface was measured easily, the detection of the cell surface transport of occludin was more difficult. If occludin was endocytosed more rapidly than transported, detection of occludin would be difficult. To test this possibility, we examined the endocytic transport of occludin using a well established biochemical assay based on cell surface biotinylation (21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar, 26Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (398) Google Scholar). As a control, a well characterized endocytosed and recycled protein, TfR, was examined. TfR expressed in BHK cells was not recovered on avidin beads without biotinylation (Fig. 1A). Endocytosed TfR increased in a time-dependent manner up to 15 min and stayed at a constant level until 120 min at 37 °C (Fig. 1A). When the endocytosis assay was performed at 18 °C, a temperature that causes the accumulation of endocytosed proteins in early/sorting endosomes (28Czekay R.P. Orlando R.A. Woodward L. Lundstrom M. Farquhar M.G. Mol. Biol. Cell. 1997; 8: 517-532Crossref PubMed Scopus (91) Google Scholar), endocytosed TfR accumulated progressively and did not reach the maximum level within 120 min (Fig. 1A). When occludin was expressed in BHK cells and subjected to cell surface biotinylation, occludin was efficiently biotinylated and isolated on avidin beads. Importantly, no occludin was detected when biotin was omitted (Fig. 1B). If the endocytosis assay was performed at 37 °C, endocytosed occludin increased linearly up to 15 min and showed a steady level until 120 min (Fig. 1B). In contrast, endocytosed occludin at 18 °C accumulated progressively up to 120 min (Fig. 1B). These results indicated that occludin as well as TfR was indeed endocytosed in BHK cells. Because claudin-1 was not endocytosed significantly when expressed in BHK cells, occludin seemed to be selected for endocytosis (17Yamamoto Y. Nishimura N. Morimoto S. Kitamura H. Manabe S. Kanayama H. Kagawa S. Sasaki T. Biochem. Biophys. Res. Commun. 2003; 308: 270-275Crossref PubMed Scopus (40) Google Scholar). To characterize further the endocytosis of occludin in BHK cells, we performed the endocytosis assay in K+-free media, a technique that has been shown to inhibit clathrin-dependent endocytosis of low density lipoprotein receptor and other receptors (21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar, 27Larkin J.M. Brown M.S. Goldstein J.L. Anderson R.G. Cell. 1983; 33: 273-285Abstract Full Text PDF PubMed Scopus (342) Google Scholar, 29Moya M. Dautry-Varsat A. Goud B. Louvard D. Boquet P. J. Cell Biol. 1985; 101: 548-559Crossref PubMed Scopus (212) Google Scholar, 30Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1993; 123: 89-97Crossref PubMed Scopus (85) Google Scholar). K+ depletion blocked the endocytosis of occludin to an extent comparable to that of TfR in BHK cells (Fig. 2, A and D), suggesting a clathrin-dependent endocytosis of occludin. Because the TJ-associated Rab family members, Rab3B and Rab13, are good candidates for regulators of occludin transport, we next assessed their function in the endocytosis of occludin. For this purpose, we generated dominant active mutants (Rab3B Q81L and Rab13 Q67L) that are defective in GTP hydrolysis as well as dominant negative mutants (Rab3B T36N and Rab13 T22N) that have a lower affinity for GTP than GDP. When these mutants were cotransfected with occludin into BHK cells, they were expressed at comparable levels and did not affect the expression level of occludin (Fig. 2B). Occludin was endocytosed in Rab3B T36N-, Rab3B Q81L-, Rab13 T22N-, or Rab13 Q67L-transfected cells as efficiently as in empty vector (mock)-transfected cells (Fig. 2, C and D). These observations indicate that occludin is endocytosed from the cell surface in a Rab3B/Rab13-independent manner in BHK cells. Endocytosed Occludin Is Recycled Continuously Back to the Cell Surface in BHK Cells—The saturable and progressive accumulation of endocytosed occludin at 37 and 18 °C, respectively, indicates recycling of endocytosed occludin back to the cell surface in BHK cells. To examine the recycling of endocytosed occludin, we performed a biochemical recycling assay, in which a decrease of biotinylated cargo molecules represents their recycling back to the cell surface. When endocytosed TfR was allowed to be recycled back to the cell surface, the amount of biotinylated TfR decreased in a time-dependent manner as expected (Fig. 3). Like TfR, endocytosed and biotinylated occludin was diminished up to 5 min (Fig. 3). This clearly demonstrates that endocytosed occludin is indeed recycled back to the cell surface in BHK cells. Rab13 Q67L Mutant Inhibits Recycling of Endocytosed Occludin, but Not TfR, Back to the Cell Surface in BHK Cells—To investigate further the recycling of occludin in BHK cells, the recycling assay was performed in the presence of BAF, a compound that inhibits the recycling of endocytosed proteins back to the cell surface by interfering with endosomal acidification (31Presley J.F. Mayor S. McGraw T.E. Dunn K.W. Maxfield F.R. J. Biol. Chem. 1997; 272: 13929-13936Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Consistent with a previous report, the recycling of TfR in the presence of BAF was inhibited compared with that in control media (21Le T.L. Yap A.S. Stow J.L. J. Cell Biol. 1999; 146: 219-232Crossref PubMed Scopus (484) Google Scholar) (Fig. 4A). BAF restrained the recycling of occludin to an extent comparable to that of TfR (Fig. 4, A and C), revealing that endocytosed occludin is indeed recycled back to the cell surface in BHK cells. Next, we examined the effect of Rab3B and Rab13 mutants on the recycling of occludin. For this purpose, the recycling of occludin was analyzed in BHK cells cotransfected with occludin and mock, Rab3B T36N, Rab3B Q81L, Rab13 T22N, or Rab13 Q67L. Occludin was recycled in Rab3B T36N- and Rab3B Q81L-transfected cells as efficiently as in mock-transfected cells, indicating that occludin is recycled in a Rab3B-independent manner (Fig. 4, B and C). In contrast, the recycling of occludin was impaired in Rab13 Q67L-transfected cells but not in Rab13 T22N-transfected cells, compared with mock-transfected cells (Fig. 4, B and C). These results demonstrate that occludin is recycled back to the cell surface via a Rab13-dependent pathway in BHK cells. The question naturally arises as to whether the Rab13-dependent recycling pathway is specific for occludin. To address this question, we examined the endocytosis and recycling of TfR in BHK cells cotransfected with TfR and Rab13 Q67L. TfR was endocytosed in Rab13 Q67L-transfected cells as efficiently as in mock-transfected cells. In contrast to occludin, the recycling of TfR was not affected by the presence of the Rab13 Q67L mutant (Fig. 5, A and B). These results demonstrate that Rab13 directs a specific endocytic recycling pathway for occludin, but not for TfR, in BHK cells.Fig. 4Rab13-dependent recycling of occludin in BHK cells. A, BHK cells expressing TfR or occludin were subjected to BAF treatment and recycling assay as in Fig. 3. After the endocytosis reaction, BAF treatment was performed by preincubating on ice for 30 min and incubating at 37 °C for 5 min in the presence of 1 μm BAF to allow the recycling reaction. B, BHK cells coexpressing occludin and mock, Rab3B T36N, Rab3B Q81L, Rab13 T22N, or Rab13 Q67L were subjected to recycling assay as in A. C, the effects of BAF treatment, Rab3B mutants, and Rab13 mutants on recycling of occludin were quantitated. Recycled proteins were expressed as the percentage of endocytosed proteins. The data shown in C are the means ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2K+-dependent endocytosis of occludin in BHK cells. A, BHK cells expressing TfR or occludin were subjected to K+ depletion and endocytosis assay as in Fig. 1A. B, BHK cells coexpressing occludin and mock, Rab3B T36N, Rab3B Q81L, Rab13 T22N, or Rab13 Q67L were subjected to immunoblot to determine the expression level of occludin

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