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• W2011970960 abstract "Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells. We have investigated the association of Rab11 family interacting protein 2 (Rab11-FIP2) with myosin Vb as an adapter protein between Rab11a and myosin Vb. Immunofluorescence studies indicated a colocalization of endogenous Rab11-FIP2 with green fluorescent protein-myosin Vb tail overexpressed in Madin-Darby canine kidney (MDCK) cells. Yeast two hybrid assays showed that amino acids 129–356 of Rab11-FIP2 were important for binding to myosin Vb tail.In vitro association assays and co-transfection experiments in both MDCK and HeLa cells confirmed this result but further refined the binding site to amino acids 129–290 of Rab11-FIP2. Like myosin Vb, functional studies indicated that Rab11-FIP2 is also important for normal plasma membrane recycling. Green fluorescent protein-Rab11-FIP2 (129–512), which lacks its amino-terminal C2 domain, functioned as a dominant negative acting truncation that caused accumulation of Rab11a and disrupted IgA trafficking in MDCK cells and transferrin trafficking in HeLa cells. The ternary association of myosin Vb and Rab11-FIP2 with Rab11a suggests that a multimeric protein complex is involved in vesicle trafficking through plasma membrane recycling systems. Plasma membrane recycling is an important process necessary for maintaining membrane composition. The motor protein myosin Vb regulates plasma membrane recycling through its association with Rab11a. Overexpression of the tail of myosin Vb disrupts trafficking out of plasma membrane recycling systems and leads to the accumulation of Rab11a in both polarized and non-polarized cells. We have investigated the association of Rab11 family interacting protein 2 (Rab11-FIP2) with myosin Vb as an adapter protein between Rab11a and myosin Vb. Immunofluorescence studies indicated a colocalization of endogenous Rab11-FIP2 with green fluorescent protein-myosin Vb tail overexpressed in Madin-Darby canine kidney (MDCK) cells. Yeast two hybrid assays showed that amino acids 129–356 of Rab11-FIP2 were important for binding to myosin Vb tail.In vitro association assays and co-transfection experiments in both MDCK and HeLa cells confirmed this result but further refined the binding site to amino acids 129–290 of Rab11-FIP2. Like myosin Vb, functional studies indicated that Rab11-FIP2 is also important for normal plasma membrane recycling. Green fluorescent protein-Rab11-FIP2 (129–512), which lacks its amino-terminal C2 domain, functioned as a dominant negative acting truncation that caused accumulation of Rab11a and disrupted IgA trafficking in MDCK cells and transferrin trafficking in HeLa cells. The ternary association of myosin Vb and Rab11-FIP2 with Rab11a suggests that a multimeric protein complex is involved in vesicle trafficking through plasma membrane recycling systems. Madin-Darby canine kidney polymeric IgA receptor Rab11 family interacting protein Rab coupling protein green fluorescent protein glutathione S-transferase Plasma membrane recycling is an essential process in the maintenance and regulation of the normal complement of membrane pumps, channels, and receptors in mammalian cells. Although the handling of individual cargo proteins may vary among cell types, a number of studies over the past several years have established a central role for the small GTPases Rab11a and Rab25 in plasma membrane recycling. In non-polarized cells, transferrin receptor recycles through a perinuclear recycling system dependent on Rab11a (1Ullrich O. Reinsch S. Urbe S. Zerial M. Parton R.G. J. Cell Biol. 1996; 135: 913-924Crossref PubMed Scopus (1084) Google Scholar, 2Green E.G. Ramm E. Riley N.M. Spiro D.J. Goldenring J.R. Wessling-Resnick M. Biochem. Biophys. Res. Commun. 1997; 239: 612-616Crossref PubMed Scopus (88) Google Scholar, 3Ren X., Xu, G. Zeng J., De Lemos-Chiarandini C. Adesnik M. Sabatini D.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6187-6192Crossref PubMed Scopus (397) Google Scholar). Rab25 is specifically expressed in epithelial cells (4Goldenring J.R. Shen K.R. Vaughan H.D. Modlin I.M. J. Biol. Chem. 1993; 268: 18419-18422Abstract Full Text PDF PubMed Google Scholar). In polarized MDCK1 cells, Rab11a and Rab25 regulate both transcytosis and apical recycling of the polymeric IgA receptor (pIgA-R) through the apical recycling system (5Casanova J.E. Wang X. Kumar R. Bhartur S.G. Navarre J. Woodrum J.E. Ray G.S. Goldenring J.R. Mol. Biol. Cell. 1999; 10: 47-61Crossref PubMed Scopus (347) Google Scholar, 6Wang X. Kumar R. Navarre J. Casanova J.E. Goldenring J.R. J. Biol. Chem. 2000; 275: 29138-29146Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Recently, several groups have reported proteins that interact with Rab11a and its related family members Rab11b and Rab25. Rab11-binding protein/Rabphilin 11 associates with GTP-bound Rab11a and colocalizes with Rab11a (7Zeng X. Ren M. Gravotta D., De Lemos-Chiarandini C. Lui M. Erdjument-Bromage H. Tempst P., Xu, G. Shen T.H. Morimoto T. Adesnick M. Sabatini D.D. Proc. Natl. Acad. Sci., U. S. A. 1999; 96: 2840-2845Crossref PubMed Scopus (70) Google Scholar, 8Mammoto A. Ohtsuka T. Hotta I. Sasaki T. Takai Y. J. Biol. Chem. 1999; 274: 25517-25524Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The pp75/Rab11 interacting protein (Rip11) localized with Myc-tagged Rab11a in rat kidney cells and associated with IgA trafficking in MDCK cells (9Prekeris R. Klumperman J. Scheller R.H. Mol. Cell. 2000; 6: 1437-1448Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The pp75/Rip11 protein is a member of a larger group of Rab11 family interacting proteins (Rab11-FIPs). Rab11-FIP1, Rab11-FIP2, Rab11-FIP3, Rab11-FIP4, Rab coupling protein (RCP), and pp75/Rip11 all interact with Rab11a through a conserved carboxyl-terminal amphipathic α-helical domain (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 11Prekeris R. Davies J.M. Scheller R.H. J. Biol. Chem. 2001; 276: 38966-38970Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 292: 909-915Crossref PubMed Scopus (67) Google Scholar, 13Lindsay A.J. Hendrick A.G. Cantalupo G. Senic-Matuglia F. Goud B. Bucci C. McCaffrey M.W. J. Biol. Chem. 2002; 277: 12190-12199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). McCaffrey and colleagues (13Lindsay A.J. Hendrick A.G. Cantalupo G. Senic-Matuglia F. Goud B. Bucci C. McCaffrey M.W. J. Biol. Chem. 2002; 277: 12190-12199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) have also reported that RCP can interact with Rab4. In addition to the Rab11 binding region, Rab11-FIP2, RCP, and pp75/Rip11 all contain an amino-terminal C2 domain (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 11Prekeris R. Davies J.M. Scheller R.H. J. Biol. Chem. 2001; 276: 38966-38970Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 292: 909-915Crossref PubMed Scopus (67) Google Scholar, 13Lindsay A.J. Hendrick A.G. Cantalupo G. Senic-Matuglia F. Goud B. Bucci C. McCaffrey M.W. J. Biol. Chem. 2002; 277: 12190-12199Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). In parietal cells, MDCK cells, and HeLa cells, multiple Rab11-FIP proteins are present spatially within populations of Rab11a-containing recycling vesicles (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Finally, we have also reported that the actin motor protein myosin Vb associates specifically with Rab11 family GTPases (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). Myosin Vb immunoreactivity was present on Rab11a-containing recycling vesicles and GFP-myosin Vb targeted to recycling system vesicles in both HeLa and MDCK cells (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). Importantly, the overexpression of the tail of myosin Vb lacking a motor domain caused accumulation of Rab11a in pericentriolar vesicle aggregates and strongly inhibited trafficking out of the recycling system in both HeLa and MDCK cells (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). Thus, GFP-myosin Vb transfection blocked exit of transferrin from the recycling system vesicles in HeLa cells and pIgA-R from the apical recycling system in MDCK cells. All of these results indicated that myosin Vb was the motor protein responsible for transit out of plasma membrane recycling systems. Myosin Vb is a member of the class V myosins (myosin Va/dilute, myosin Vb/myr6, and myosin Vc) that promote transit along the actin cytoskeleton and appear to associate with distinct Rab GTPases. Although the Rab protein partner for myosin Vc remains obscure (15Rodriguez O.C. Cheney R.E. J. Cell Sci. 2002; 115: 991-1004Crossref PubMed Google Scholar), recent investigations have demonstrated the association of myosin Va with vesicles containing Rab27a (17Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). Myosin Va organizes the distribution and transport of Rab27a-containing melanosomes in melanocytes (16Hume A.N. Collinson L.M. Rapak A. Gomes A.Q. Hopkins C.R. Seabra M.C. J. Cell Biol. 2001; 152: 795-808Crossref PubMed Scopus (274) Google Scholar, 17Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar). Mutations in Rab27a or myosin Va lead to neurological abnormalities and the inability to recruit melanosomes in both rodent models and humans (18Wilson S.M. Yip R. Swing D.A. O'Sullivan T.N. Zhang Y. Novak E.K. Swank R.T. Russell L.B. Copeland N.G. Jenkins N.A. Proc. Natl. Acad. Sci. 2000; 97: 7933-7938Crossref PubMed Scopus (339) Google Scholar, 19Schuster F. Stachel D.K. Schmid I. Baumeister F.A. Graubner U.B. Weiss M. Haas R.J. Belohradsky B.H. Bone Marrow Transplant. 2001; 28: 409-412Crossref PubMed Scopus (35) Google Scholar, 20Pastural E. Barrat F.J. Dufourcq-Lagelouse R. Certain S. Sanal O. Jabado N. Seger R. Griscelli C. Fischer A. de Saint Basile G. Nat. Genet. 1997; 16: 289-292Crossref PubMed Scopus (367) Google Scholar). The bond between Rab27a and myosin Va is indirect (17Wu X. Rao K. Bowers M.B. Copeland N.G. Jenkins N.A. Hammer III, J.A. J. Cell Sci. 2001; 114: 1091-1100Crossref PubMed Google Scholar), and recent efforts have indicated that melanophilin/Slp homologue lacking C2 domains (Slac2-a) functions as the receptor recruited by Rab27a for myosin Va binding (21Fukada M. Kuroda T.S. Mikoshiba K. J. Biol. Chem. 2002; 277: 12432-12436Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 22Hume A.N. Collinson L.M. Hopkins C.R. Strom M. Barral D.C. Bossi G. Griffiths G.M. Seabra M.C. Traffic. 2002; 3: 193-202Crossref PubMed Scopus (133) Google Scholar, 23Wu X.S. Rao K. Zhang H Wang F. Sellers J.R. Matesic L.E. Copeland N.G. Jenkins N.A. Hammer III, J.A. Nat. Cell. Biol. 2002; 4: 271-278Crossref PubMed Scopus (383) Google Scholar). Rab11-FIP2 localized with Rab11a in MDCK and HeLa cells and associated with Rab11a in yeast two hybrid assays (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). In vitro, [γ-35S]GTP Rab11a bound directly to recombinant Rab11-FIP2 (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Rab11-FIP2 co-enriched with Rab11a in rabbit gastric membrane fractions and translocated with Rab11a to the canaliculus in stimulated parietal cells (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Recent studies from McCaffrey and colleagues (12Wallace D.M. Lindsay A.J. Hendrick A.G. McCaffrey M.W. Biochem. Biophys. Res. Commun. 2002; 292: 909-915Crossref PubMed Scopus (67) Google Scholar) have suggested that Rab11-FIP2 can dimerize and may also form hetero-oligomers with other Rab11-FIP family members. Importantly, our original yeast two hybrid studies indicated that Rab11-FIP2 could interact with the tail of myosin Vb (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). We hypothesized that Rab11-FIP2 may contribute to anchoring of Rab11a-positive vesicles to myosin Vb. We now report that yeast two hybrid analysis, immunolocalization experiments, and in vitro binding studies all confirm the direct association of Rab11-FIP2 with myosin Vb. In trafficking studies, a truncation of Rab11-FIP2 lacking the amino-terminal C2-domain inhibited plasma membrane recycling in both HeLa and MDCK cells. These studies show that Rab11-FIP2 is a receptor/adapter protein linking myosin Vb to Rab11a and regulating the movement of vesicle cargo through the recycling endosome. Yeast two hybrid binary assays were conducted as described previously (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Briefly, truncations of Rab11-FIP2 were amplified by PCR adding an EcoRI site to the 5′ end and a SalI site to the 3′end. PCR amplification products were then digested with EcoRI andSalI (New England Biolabs) and cloned into pBD-Gal-(CaM) (Stratagene) vector utilizing T4 DNA ligase (New England Biolabs). Plasmids were confirmed with automated sequencing (Molecular Biological Core Facility, Medical College of Georgia). Rab11-FIP2 truncations were then cotransfected with either pAD-myosin Vb tail or pAD empty vector as a control into the yeast strain Y190. Yeast were allowed to grow for 3 days at 30 °C and then analyzed via β-galactosidase assay. A positive result represented blue color within 4 h. Recombinant GST-myosin Vb tail attached to glutathione-Sepharose beads and recombinant His-Rab11-FIP2, His-Rab11-FIP2 (129–356), His-Rab11-FIP2 (129–290), and His-Rab11-FIP2 (191–290) were made as described previously (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 24Goldenring J.R. Smith J. Vaughan H.D. Cameron P. Hawkins W. Navarre J. Am. J. Physiol. 1996; 270: G515-G525PubMed Google Scholar). Briefly, myosin Vb tail was amplified with SalI sites added to both ends. The sample was cut with SalI, cloned into pGEX5x-1, and confirmed for sequence accuracy. Myosin Vb tail in pGEX5x-1 and pGEX5x-1 empty vector were transformed into BL21pLysS competent cells (Promega), and recombinant protein was produced following induction with isopropyl-1-thio-β-d-galactopyranoside (1 mm) for 3 h. GST and GST-myosin Vb tail beads were stored in phosphate-buffered saline with protease inhibitor mixture (Sigma) at 4 °C. Rab11-FIP2 and Rab11-FIP2 truncations were PCR amplified and cloned into pET30a (Novagen) for His-tagged recombinant protein production. For a single pull-down reaction, 50 μl of GST and GST-myosin Vb tail beads were blocked for 2 h in buffer containing 1% bovine serum albumin, 50 mm Tris, pH 7.5, 0.5 mmMgCl2, 1 mm EGTA, 10 mmdithiothreitol, 1:200 protease inhibitor mixture. Approximately 2 μg of His-Rab11-FIP2, His-Rab11-FIP2 (129–356), His-Rab11-FIP2 (129–290), or His-Rab11-FIP2 (191–290) were combined with either GST or GST-myosin Vb tail beads. Samples were incubated with gentle shaking for 1 h at room temperature. Supernatants were removed, and remaining His-tagged protein not bound to GST or GST-myosin Vb tail beads was rescued utilizing His-bind resin (Novagen). The GST and GST-myosin Vb tail beads (pellets) were washed in reaction buffer without bovine serum albumin. The supernatant and pellet beads were resuspended in SDS loading buffer and loaded onto SDS-PAGE gels. Gels were transferred to Immobilon-P membrane (Millipore), and Western blots were performed. Because the His-tagged proteins contained an amino-terminal S protein tag, proteins were detected utilizing an S protein-horseradish peroxidase-conjugated secondary antibody (Novagen). Transient transfections were performed utilizing Effectene transfection reagent (Qiagen). Immunostaining was performed as described previously (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Imaging of dual transfection experiments with GFP-Rab11-FIP2 truncations and DsRed myosin Vb tail was performed utilizing a Zeiss LSM-510 confocal fluorescence microscope. All other images were obtained with a Molecular Dynamics confocal microscope. The GFP-Rab11-FIP2 (129–512) sequence was digested from pEGFP-Rab11-FIP2 (129–512) withNheI and SalI and cloned into the tetracycline responsive plasmid, pTRE2 (Clontech). Utilizing Effectene, GFP-Rab11-FIP2 (129–512) in pTRE2 was then transfected dually with pCB7 vector into an MDCK tetracycline repressible cell line (T23) (25Mostov K.E. Deitcher D.L. Cell. 1986; 46: 613-621Abstract Full Text PDF PubMed Scopus (213) Google Scholar) and plated onto 110-mm dishes. Cell medium was supplemented with G418 (0.5 mg/ml) and doxycycline (5 ng/ml). Two days after transfection, cells were trypsinized and replated in serial dilutions on 110-mm plates. After 7 days, single colonies were picked and transferred to 24-well plates. After 7 more days cells were trypsinized and transferred to 12-well plates in the absence or presence of doxycycline. Cell lines expressing GFP-Rab11-FIP2 (129–512) were passaged to six-well plates, T25 flasks, and finally, T75 flasks. GFP-Rab11-FIP2 (129–512) tetracycline repressible cell lines were tested for clonality via imaging and for expression levels via Western blot. IgA apical recycling and transcytosis assays were performed by modification of previously described methods (5Casanova J.E. Wang X. Kumar R. Bhartur S.G. Navarre J. Woodrum J.E. Ray G.S. Goldenring J.R. Mol. Biol. Cell. 1999; 10: 47-61Crossref PubMed Scopus (347) Google Scholar) adapted for the use of fluorescent ligand. Briefly, Alexa 546 dye was covalently attached to polymeric IgA (Alexa Fluor 546 protein labeling kit). Alexa 546-IgA (10 μg/ml) was then added to the apical surface or the basolateral surface of transwell filters containing confluent MDCK cells stably transfected with the polymeric IgA receptor and GFP-Rab11-FIP2 (129–512) in the presence or absence of doxycycline (5 ng/ml). Medium without dye was utilized for IgA trafficking assays to reduce background fluorescence. For fluorescence imaging, cells were fixed in 4% paraformaldehyde at 0 and 40 min for apical recycling assays and 0 and 60 min for transcytosis assays. For quantitative analysis, apical and basolateral supernatants and cell bodies were collected at 0 and 40 min for apical recycling and 0 and 60 min for transcytosis. Samples were suspended in SDS buffer (final concentration, 300 mm Tris, pH 7.5, 1% SDS, 20 mm EDTA, 17.5 mm sucrose) and resolved utilizing SDS-PAGE. Low fluorescence plates (Amersham Biosciences) were utilized for preparing the polyacrylamide gels. Gels were then scanned utilizing an Amersham Biosciences two-dimensional fluorescent gel scanner. Fluorescent IgA bands were quantified for pixel intensity utilizing GeneQuant software. At different time points, the quantitated amounts of IgA in the apical and basolateral media, as well as label remaining within cells, were determined and used to calculate the percent of total IgA retained in cells: [(IgAcells)/(IgAcells + IgAapical media + IgAbasolateral media)] × 100. At least three separate experiments in triplicate were conducted for each condition. Differences among groups were analyzed for statistical significance with Student's t test (InStat). Transferrin trafficking was conducted as described previously with differences noted below (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). HeLa cells containing transiently transfected GFP-Rab11-FIP2 (129–512) were utilized to assess trafficking. Alexa 546-conjugated transferrin (Molecular Probes) was loaded into the cells. For images, cells were fixed at 0 and 40 min. Cells were then stained for transferrin receptor (CD71). The uptake of transferrin into HeLa cells was quantitated by determining Alexa 546 fluorescence in transfected and non-transfected cells using digital image analysis (Metamorph). For quantitative analysis, imaging of HeLa cells was performed at ×63 using a Zeiss LSM-510 confocal fluorescence microscope capturing 2-μm optical sections. All images were obtained with identical exposure settings and were analyzed without further image processing. Integrated fluorescence in an 8-μm-diameter circular perinuclear region in 100 cells was determined for each condition. Results were expressed as a mean of arbitrary fluorescence intensity units per cell. Our previous studies had implicated myosin Vb as the motor protein for transit out of the plasma membrane recycling endosome system (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). We therefore examined the localization of endogenous Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 (pp75/Rab11 interacting protein) with overexpressed GFP-myosin Vb tail. In polarized MDCK cells, endogenous Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 co-localized with both GFP-myosin Vb tail and endogenous Rab11a (Fig. 1). The results suggest that all of these Rab11 interacting proteins may function in a contiguous vesicle recycling system ultimately dependent on myosin Vb activity. Because yeast two hybrid data has indicated an association between myosin Vb and Rab11-FIP2 (10Hales C.M. Griner R. Hobdy-Henderson K.C. Dorn M.C. Hardy D. Kumar R. Navarre J. Chan E.K.L. Lapierre L.A. Goldenring J.R. J. Biol. Chem. 2001; 276: 39067-39075Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), further yeast two hybrid assays were used to determine the myosin Vb binding site in Rab11-FIP2. Amino acids 129–356 of Rab11-FIP2 (Rab11-FIP2 (129–356)) were sufficient to maintain an association with myosin Vb tail (Fig. 2). Interestingly, when truncated from the carboxyl terminus to amino acid 465, Rab11-FIP2 would associate nonspecifically. Truncation analysis indicated that the autoactivation domain mapped to amino acids 429–465 in Rab11-FIP2 (data not shown). Similar yeast two hybrid assays were conducted in which full-length Rab11-FIP2 was tested against deletion mutants of myosin Vb tail. Deletion of either 45 amino acids of the amino terminus (myosin Vb tail (45–588)) or 32 amino acids of the carboxyl terminus (myosin Vb tail (1–553)) of the myosin Vb tail eliminated interaction with Rab11-FIP2. In contrast, we have demonstrated previously that myosin Vb tail (45–588) and myosin Vb tail (1–553) could interact with Rab11a in yeast two hybrid studies (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). To confirm the myosin Vb tail/Rab11-FIP2 association, GST fusion protein binding assays were used. Rab11-FIP2 strongly associated with GST-myosin Vb tail beads compared with little detectable interaction with GST control beads (Fig. 3). Despite the presence of protease inhibitors, His-Rab11-FIP2 was highly susceptible to proteolysis especially in supernatant fractions (data not shown). In agreement with yeast two hybrid results, Rab11-FIP2 (129–356) was also retained on GST-myosin Vb tail beads, compared with no detectable association with GST beads. (Fig. 3). Interestingly, GST binding assays indicated an association between myosin Vb tail and Rab11-FIP2 (129–290) but not the smaller fragment Rab11-FIP2 (191–290) thus narrowing the myosin Vb tail binding region. To confirm the association of myosin Vb and Rab11-FIP2 in situ, we studied the distribution of GFP-Rab11-FIP2 truncations with co-expressed DsRed-myosin Vb tail. In singly transfected MDCK cells, GFP-Rab11-FIP2 was distributed in a multi-vesiculated pattern similar to that observed for immunostaining of endogenous Rab11-FIP2 (Fig. 4 A). However, GFP-Rab11-FIP2 (129–512), which lacked the amino-terminal C2 region of Rab11-FIP2, was distributed to a more compact large punctate spot (Fig. 4 A). Other GFP-Rab11-FIP2 truncations, which did not contain the carboxyl-terminal Rab11 binding site, demonstrated a predominantly cytosolic distribution (Fig. 4 A). In MDCK cells (Fig. 4 B), both GFP-Rab11-FIP2 and GFP-Rab11-FIP2 (129–512) colocalized with co-expressed DsRed-myosin Vb tail. As a control, non-chimeric GFP alone did not colocalize with DsRed-myosin Vb tail (data not shown). Importantly, compared with their cytosolic distribution in single transfection studies (Fig. 4 A), GFP-Rab11-FIP2 (1–465), GFP-Rab11-FIP (129–356), and GFP-Rab11-FIP2 (129–290) colocalized with DsRed-myosin Vb tail in a punctate pattern. Identical results were observed with dual transfections into HeLa cells (data not shown). These studies confirmed the in vitroassociation assays. In MDCK cells stably expressing pIgA-R, apical recycling or transcytosis of IgA is trafficked through Rab11a-containing vesicles (5Casanova J.E. Wang X. Kumar R. Bhartur S.G. Navarre J. Woodrum J.E. Ray G.S. Goldenring J.R. Mol. Biol. Cell. 1999; 10: 47-61Crossref PubMed Scopus (347) Google Scholar). GFP-myosin Vb tail concentrates pIgA-R and disrupts IgA transcytosis (14Lapierre L.A. Kumar R. Hales C.M. Navarre J. Bhartur S.G. Burnette J.O. Mercer J.A. Bahler M. Goldenring J.R. Mol. Biol. Cell. 2001; 12: 1843-1857Crossref PubMed Scopus (347) Google Scholar). Thus, we sought to determine the involvement of Rab11-FIP2 in IgA trafficking. GFP-Rab11-FIP2 (129–512) was chosen for the assay, because it elicited a tight punctate distribution similar to the dominant negative acting myosin Vb tail (Fig. 4 A). A tetracycline repressible line containing GFP-Rab11-FIP2 (129–512) was created to compare trafficking in cells in the absence or presence of overexpressed GFP-Rab11-FIP2 (129–512). Fig. 5 demonstrates that polymeric IgA-R co-localized with both GFP-Rab11-FIP2 (129–512) and Rab11a in concentrated intracellular puncta in each cell. Western blots of whole cell extracts from cells grown in the presence or absence of doxycycline indicated undetectable expression levels of GFP-Rab11-FIP2 (129–512) in the presence of 5 ng/ml antibiotic (Fig. 6 B, inset).Figure 6IgA trafficking in polarized MDCK cells. A, IgA trafficking was assessed in MDCK cells stably transfected with GFP-Rab11-FIP (129–512) either without doxycycline (−DOX; expressing GFP-Rab11-FIP2 (129–512)) or with doxycycline (+DOX; not expressing GFP-Rab11-FIP2 (129–512)). Alexa 546-IgA was loaded basolaterally for IgA transcytosis. Cells were fixed at 0 and 60 min (T-0 andT-60) for imaging. Alexa 546-IgA was loaded apically for IgA apical recycling. Cells were fixed at 0 and 40 min (A-0 and A-40) for imaging. Images are look-through confocal microscopic reconstructions with Z axis projections below. White arrows indicate points of colocalization between GFP-Rab11-FIP2 (129–512) and Alexa 546-IgA.Black arrowheads on far right indicate the location of Z series. White bar in lower leftequals 5 microns. The figure is representative of three separate experiments. B, percent of IgA retained in cells (± S.E.) for IgA transcytosis at the 60-min time point. *, p = 0.013. Western blot inset shows GFP-Rab11-FIP2 (129–512) expression in cells incubated in the absence (−DOX) and presence (+DOX) of doxycycline. C, percent of IgA retained in cells (± S.E.) for IgA apical recycling at the 40-min time point. **, p = 0.007. The f" @default.
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