Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized ...

16 Pages·1998·0.81 MB·English

Rab17 Regulates Membrane Trafficking through Apical
Recycling Endosomes in Polarized Epithelial Cells
Paola Zacchi,* Harald Stenmark,‡ Robert G. Parton,(cid:105) Donata Orioli,* Filip Lim,§ Angelika Giner,*
Ira Mellman,¶ Marino Zerial,* and Carol Murphy*,**
*European Molecular Biology Laboratory, Postfach 10.2209, D-69012 Heidelberg, Germany; ‡Department of Biochemistry, the
Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway; §Centro de Biologia Molecular, Facultad de Ciencias,
Universidad Autonoma de Madrid, 28049 Cantoblanco, Spain; (cid:105)Centre for Microscopy and Microanalysis, Department of
Physiology and Pharmacology, and Centre for Molecular and Cellular Biology, University of Queensland, 4072 Brisbane,
Australia; ¶Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520; and **Laboratory
of Biological Chemistry, Medical School, University of Ioannina, 45110 Ioannina, Greece
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Abstract. A key feature of polarized epithelial cells is some that has been implicated in recycling and transcy- ed
the ability to maintain the specific biochemical compo- tosis. The localization of rab17, therefore, strengthens from
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sition of the apical and basolateral plasma membrane the proposed homology between this compartment and ttp
domains while selectively allowing transport of proteins the recycling endosome of nonpolarized cells. Basolat- ://ru
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of regulators of intracellular transport, is specifically in- meric receptor, was specifically increased in Eph4 cells /jcb
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duced during cell polarization in the developing kidney. expressing rab17 mutants defective in either GTP bind- rticle
We here examined its intracellular distribution and ing or hydrolysis. Furthermore, the mutant proteins -pd
function in both nonpolarized and polarized cells. By stimulated apical recycling of FcLR 5-27. These results f/14
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confocal immunofluorescence microscopy, rab17 colo- support a role for rab17 in regulating traffic through the /1
0
3
calized with internalized transferrin in the perinuclear apical recycling endosome, suggesting a function in po- 9
/1
2
recycling endosome of BHK-21 cells. In polarized Eph4 larized sorting in epithelial cells. 79
5
cells, rab17 associated with the apical recycling endo- 95
/2
9
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Eukaryotic cells continuously endocytose mem- bules and vesicles in the pericentriolar region of the cell, y gue
brane proteins, lipids, and solutes. After internal- whose organization is maintained by microtubules (Hop- st on
2
ization, these molecules are selectively recycled kins, 1983a; Yamashiro et al., 1983; Yamashiro and Maxfield, 2 F
e
back to the cell surface or routed to the degradative path- 1987; Ghosh et al., 1994). These elements represent the b
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way. All these sorting events are mediated by a series of so-called recycling endosome (Gruenberg and Maxfield, ary 2
heterogeneous and highly dynamic endosomal compart- 1995) or para-Golgi recycling compartment (Yamashiro 0
2
3
ments (for reviews see Watts and Marsh, 1992; Gruenberg and Maxfield, 1984). Transit through the recycling endo-
and Maxfield, 1995; Mellman, 1996). Separation of recy- some proceeds with slower kinetics compared with trans-
cling receptors from lysosomally directed proteins takes port through the early endosome (Yamashiro and Maxfield,
place within the early endosomes, also named sorting en- 1984; Hopkins et al., 1994). The definition of boundaries
dosomes, located in the periphery of the cell (Gruenberg between these extremely plastic endocytic structures is
and Maxfield, 1995). From here, recycling molecules can controversial, and in some cell types, endosomes appear to
be rapidly transported back to the plasma membrane and, form a continuous intricate network spread throughout
at least in part, transit through a separate network of tu- the cytoplasm (Hopkins et al., 1990). Nevertheless, the as-
sociation of the small GTPases rab4 (van der Sluijs et al.,
1992a; Daro et al., 1996) and rab5 (Chavrier et al., 1990;
Address all correspondence to Carol Murphy, Laboratory of Biological Bucci et al., 1992) with sorting endosomes and rab11 with
Chemistry, Medical School, University of Ioannina, 45110 Ioannina, recycling endosomes (Ullrich et al., 1996), provides sup-
Greece. Tel.: [phone]. Fax: [phone]. E-mail: [email protected] port to the existence of functionally and biochemically dis-
Marino Zerial, European Molecular Biology Laboratory, Postfach
tinct endosomal compartments.
10.2209, 69012 Heidelberg, Germany. Tel.: [phone]. Fax: 49 6221
387 306. E-mail: [email protected] The organization of the endocytic pathway acquires an
ª The Rockefeller University Press, 0021-9525/98/03/1039/15 $2.00
The Journal of Cell Biology, Volume 140, Number 5, March 9, 1998 1039–1053
http://www.jcb.org 1039
This document was created with FrameMaker 4.0.4
additional level of complexity in polarized epithelial cells, be modified to function in polarized sorting through the
which have to maintain a characteristic spatial and func- expression of new regulatory molecules.
tional asymmetry (Simons and Wandinger-Ness, 1990; Nel- A small GTPase of the rab family, rab17, has been pre-
son, 1992; Rodriguez-Boulan and Powell, 1992; Mostov et viously identified to be induced during cell polarization
al., 1992; Mellman, 1996). The presence of two distinct (Lütcke et al., 1993). In developing kidney, rab17 is absent
plasma membrane domains requires separate endocytic from the mesenchymal precursors, but is expressed upon
and biosynthetic pathways and a communication route their differentiation into polarized epithelial cells. Rab17
that allows transport from one cell surface to the opposite was localized to the so-called apical dense tubules, vesicu-
one, i.e., transcytosis (Rodman et al., 1990; Mostov et al., lar and tubular structures underlying the apical plasma
1992; Mostov, 1994). Studies in polarized MDCK cells membrane which, based on studies of kidney proximal tu-
have suggested that fluid-phase markers, internalized from bule cells, have been implicated in transcytosis and mem-
the apical or basolateral plasma membrane domain, accu- brane recycling to the apical surface (Christensen, 1982;
mulate in distinct apical and basolateral early endosomes Nielsen et al., 1985). Given the important role played by
before meeting in late endosomes (Bomsel et al., 1989; rab proteins in the regulation of membrane traffic (for re-
Parton et al., 1989; Fujita et al., 1990). However, recent ev- views see Pfeffer, 1994; Nuoffer and Balch, 1994; Novick
idence suggested the existence of a single common endo- and Zerial, 1997), the induction of rab17 at the onset of
somal compartment that receives markers internalized cell polarization may reflect a function in polarized mem-
from both apical and basolateral membranes. Electron mi- brane transport.
croscopy studies in polarized Caco2 cells showed that In the present study we have analyzed the intracellular
transferrin (Tf)1, internalized from the basolateral side, distribution of rab17 both in nonpolarized BHK-21 cells
was delivered to an apically located endocytic compart- and in polarized epithelial Eph4 cells (Reichmann et al.,
ment accessible to apical membrane markers (Hughson 1989; Reichmann et al., 1992). We have found that rab17
and Hopkins, 1990; Knight et al., 1995). In MDCK cells, associates with the perinuclear recycling endosome when
basolaterally internalized polymeric IgA receptor (pIgR) ectopically expressed in nonpolarized cells and with the
is first delivered to basolateral endosomes, and then apical recycling compartment in fully polarized epithelial
reaches an apical tubular endosomal compartment before cells. We have investigated the role of rab17 in receptor
being delivered to the apical plasma membrane (Barroso trafficking in polarized Eph4 cells expressing wild type
and Sztul, 1994; Apodaca et al., 1994). Altogether these re- rab17 or dominant interfering mutants in an inducible
sults suggest that, within this structure named the apical manner. Our results implicate rab17 in the control of
recycling compartment (Barroso and Sztul, 1994; Apodaca transport through the apical recycling endosome and sug-
et al., 1994), recycling receptors and transcytosing mole- gest that this GTPase is induced during the generation of
cules are sorted in a polarized fashion. The extent to which cell polarity to link the basolateral and apical endocytic
apical and basolateral endosomes contribute to polarized pathways, and to regulate polarized membrane sorting.
sorting, and the exact nature of the apical recycling com-
partment and its relationship with other portions of the
Materials and Methods
endocytic pathway still remain to be elucidated. Interest-
ingly, however, the apical recycling endosome shares strik-
Cell Culture and Antibodies
ing similarities with the recycling endosome described in
nonpolarized cells: it exhibits a tubular morphology, it is Eph4 cells were obtained from H. Beug (Research Institute of Molecular
clustered around the microtubule organizing center, and it Pathology, Vienna, Austria) and were grown in DME supplemented with
is reached by membrane markers, such as transferrin re- 2 mM glutamine, 10% heat-inactivated FCS, 100 U/ml penicillin, and 10
(cid:109)g/ml streptomycin. For growth on polycarbonate filters, 0.4 (cid:109)m pore di-
ceptor (TR; Apodaca et al., 1994), although Barroso and
ameter, 12 mm filter diameter (Transwell; Costar Corp., Cambridge, MA),
Sztul (1994) failed to detect Tf in this compartment. It is
cells were trypsinized, a single cell suspension was made, and seeded at a
also poorly labeled by fluid phase markers, presumably density of 2.5 (cid:51) 105 per filter. Medium was placed on both surfaces, 0.5 ml
due to sorting in the early endosomes. These similarities apically and 1.5 ml basolaterally, and was changed daily.
therefore suggest that the apical recycling endosome may BHK-21 cells were cultured in Glasgow MEM supplemented with 5%
heat-inactivated FCS, 10% tryptose phosphate, 2 mM glutamine, 100 U/ml
be equivalent to the recycling endosome of nonpolarized penicillin, and 10 (cid:109)g/ml streptomycin. All media and reagents for cell cul-
cells. ture were purchased from GIBCO BRL (Eggenheim, Germany).
How would this compartment function in polarized sort- Zo-1 antibody R26.4C was obtained from Developmental Studies Hy-
ing in epithelial cells? One possibility is that epithelial cells bridoma Bank (University of Iowa, Iowa City, IA). The antibody for the
FcLR 5-27 chimeric receptor was purified from 2.4G2 hybridoma superna-
would not require any additional components of the traf-
tant (Unkeless, 1979) by ammonium sulfate precipitation. Briefly, the
ficking machinery compared with nonpolarized cells. Fi-
2.4G2 hybridoma supernatant was adjusted to 55% saturation with
broblasts already possess elements of the apical and baso- (NH)SO (Sigma Chemical Co., St. Louis, MO). The precipitate col-
42 4
lateral biosynthetic routes (Musch et al., 1996; Yoshimori lected by centrifugation was dissolved in HO (20 ml of HO per liter of
2 2
et al., 1996), supporting the notion that at least some com- supernatant) and dialyzed against protein G binding buffer (10 mM
Na2HPO/NaHPO buffer, pH 7.0, 10 mM EDTA, 150 mM NaCl). A
ponents involved in cell polarization may exist in nonpo- 4 2 4
preswollen gamma bind plus sepharose column (Pharmacia Diagnostics
larized cells. Alternatively, the recycling endosome could AB, Uppsala, Sweden) was equilibrated with binding buffer, the sample
was applied and bound antibodies were eluted with 0.5 M acetic acid/NH,
3
pH 2.5. 1 ml fractions were collected into tubes containing the appropriate
volume of 2 M Tris base.
1. Abbreviations used in this paper: ECL, electrochemiluminescence; hTR, An antibody was raised against a synthetic peptide corresponding to
human transferrin receptor; IFN, interferon; RT, room temperature; Tf, rab17 NH-terminal amino acids NH-MAQAAGLPQASTASQPK-COOH.
2 2
transferrin; TR, transferrin receptor; WT, wild type. The 49-1 antibody was raised against the entire rab17 protein. Rab17 wild
The Journal of Cell Biology, Volume 140, 1998 1040
type (WT) was cloned into pRsetA vector (Invitrogen, Carlsbad, CA), Alto, CA) as a selectable marker. The colonies were selected in 800 (cid:109)g/ml
transformed into Bl21 DE3 cells, and the expressed protein was purified G418 and 1 (cid:109)g/ml puromycin (Sigma Chemical Co.). Clones were
by nickel agarose affinity chromatography and used to immunize rabbits. screened by indirect immunofluorescence for both rab17 (antibody 49-1)
For immunofluorescence, the serum was affinity purified using nitrocellu- and FcLR 5-27 (monoclonal 2.4G2 antibody). The above polarity checks
lose strips of the recombinant protein. For Western blot analysis the crude were then carried out. Clones were expanded and frozen; all assays were
serum was used. The antibody against cellubrevin was kindly provided by performed on early passage cells. Levels of rab17 before and after induc-
P. De Camilli (Yale University, New Haven, CT; Galli et al., 1994). All tion with interferon were also analyzed by Western blot analysis.
secondary antibodies were obtained from Dianova (Hamburg, Germany). Eph4 cells were seeded onto six-well plastic dishes and grown to 80%
confluence. One well was induced with interferon and the other left unin-
Preparation and Biotinylation of the Fab Fragment duced as a control. After the induction period, the cells were washed in
PBS and lysed in 200 (cid:109)l PBS, 1% SDS, 0.3 mM PMSF (Sigma Chemical
The purified 2.4G2 IgG was dialyzed against papain buffer (0.1 M NaAce- Co.), sonicated, boiled for 10 min, and resuspended in loading buffer. Pro-
tate, pH 5.5). 6 mg of 2.4G2 IgG were digested with insoluble enzyme as tein content was assayed using the BCA protein assay reagents (Pierce)
described by the manufacturer (Sigma Chemical Co). Fab fragments were and BSA as a standard. 20 (cid:109)g total protein was loaded onto a 12% SDS-
biotinylated with NHS-LC-biotin or NHS-SS-biotin (Pierce, Rockford, IL) PAGE, blotted, and hybridized with a rabbit polyclonal antibody (49-1).
after the protocol described by the manufacturer (ImmunoPure NHS-
LC-biotinylation Kit; Pierce).
Localization of Rab17 WT Protein in Polarized
Colocalization of Rab17 and Internalized Eph4 Cells
FITC-Labeled Transferrin in Nonpolarized Cells
Eph4 cells, harboring Mx-rab17 WT construct, were grown on filter sup-
DNAs encoding rab17 WT and the human transferrin receptor (Zerial et al., ports; rab17 was induced with interferon and filters were rinsed in PBS(cid:49)
1u9n8d6e)r wtheer eT 7c lopnroemd ointetor. pBGHEKM c-e1ll sv ewcetroer t(rPyrposmineizgead C2o4 rhp .b, eMfoardei storann, sWfeIc)- a(PldBeSh ywdieth f o1 rm 1M5 mCianC al2t arnodo m1 m teMm Mpegr aCtul2r)e a (t R37T(cid:56))C, ,p feixremde ianb i3l%ize dp airna f0o.r1m%- Do
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tion and seeded onto 11 mm coverslips. Cell were infected with T7 RNA Triton X-100 for 4 min, and quenched in 50 mM ammonium chloride for nlo
polymerase recombinant vaccinia virus (Stenmark et al., 1995) and 15 min. Filters were then cut from the supports, blocked in 10% FCS for ad
e
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ao(cid:109)tfg 3/sme7r(cid:56)lCu Fm. ICT toChve-el acrboslevipleesrd swl itperarse nw stfehererenr i inrni cn(uUsbeldalrt iiecndh P ienBt Saa lah.,n u1dm9 9fiid3xi)ef idfeo dirn c1 3h5%a, m3 0pb,a eorrar wf6oi0rt mhm a5iln0- 3dco0o nnmckeienny ta ratan RttiioT–nr i anob f1b 0 imt. 1Ig g(cid:109)/Gmg./l m NDlu N(cSaleisgie mw freaer eCe- hv ReismNuaiaclsiaezleA Cd (owS.)iitg hfmo pra r 1oC5ph imedmiiunim caa til o RCdToid.,) ea. afFttei lar- http://rup
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focal microscopy. theRn aabn1a7ly lzoecda lbizya tcioonnf owcaasl amlsiocr aossscaoypeyd. by immunoelectron microscopy on rg/jcb/a
GExenpreersastiioonn Cofo MnsXtr-uRcatsb 17 and MX-FcLDL5-27 psclraisbtiecd g(rCohwanv rEiepr he4t acle.,l l1 9li9n0e) .harboring Mx-rab17 WT as previously de- rticle-pd
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The Mx promoter was excised as a 1.7-kb fragment EcoRI-BamHI from 0/5
pMXhGH (Hug et al., 1988) and inserted into p163/7 (Woodroofe et al., Colocalization of Rab17 and Internalized FITC-labeled /1
0
1992), from which the H2 promoter had been excised. Mx-rab17 con- Transferrin in Filter-grown Eph4 Cells 39
/1
structs were generated by PCR. A consensus Kozak site (Kozak, 1987) 2
7
was introduced 5(cid:57) to the ATG, and NheI sites were introduced to clone Eph4 cells harboring Mx-rab17 WT were grown on filter supports, and ex- 95
9
the open reading frame into the Mx expression cassette. The mutations in pression of rab17 was induced by the addition of interferon for 12 h. The 5/2
the coding region of rab17 to generate rab17Q77L and rab17N132I were coding region of the human transferrin receptor (hTR) was cloned into 93
9
ceGat TrrAaielC.d -o3(1(cid:57)u 9t 9(b0ry)a bP1uC7sRQin-7gb7 aLsoe)l dig aomnsu dt 5a(cid:57)g5-eG(cid:57)n-GGesCGisT AaGcCcGAoTrCdCAinGGgG tCoCC AtThTeT CCmACeAtThGAodGA oACf GGLAGanA-d3(cid:57)-t tw1h9ae9s 6 at)hm aepnnld ipc oaucnske vadeg tceotdo irinn fpteoHc tHS MVSV x(- Grpaaebrll1tei7cr l eWasn Tads- t Bprarrneevasfikeoecuftsieeldyld dE, e1ps9hc8r4i8 b)ce.e dlpl sH( LgSirVmow- enht TaoRln., 8.pdf by g
(rab17N132I). filter supports (Murphy et al., 1997). Cells were infected in serum-free me- ue
The FcLR 5-27 chimeric receptor (Matter et al., 1993)–coding region dium (to deplete transferrin) for 4 h, washed briefly at 37(cid:56)C, and 50 (cid:109)g/ml st o
fragment EcoRI-XbaI was excised after changing by PCR the consensus FITC-labeled transferrin was added to the basolateral surface for 90 min n 2
Kozak site 5(cid:57) to the upstream methionine, using oligos Fc-KOZ (5(cid:57)-GAT at 37(cid:56)C in a humidified chamber. Cells were then washed briefly, fixed in 2 F
XiCnGh EoA cpoArRTimIT-eXCrb G(a5CI(cid:57) C-sCiGtGeCsG CoGfG GpCGGCECAMTTC-G1G GvAeGcGAtoTArT T(TPCGrCoAmTCGeCgCaAC CCGoATrp-T3.,C(cid:57) )M-, 3aa(cid:57))nd dis aoinnnds, e WrFteIc)d-. 3rEe%psch ep4n accreeal flaoss r wmdeeasrlcedr eigbhreyoddw ebn,e aloonnwd p.r Ialanbs 1tthi7ce ,w caaanssd ev iohsfuu tmahliaezn ee dxh pobelyor ii-mnTdef inr(teS csight moimwam nC uinhn eFomfigliu.c oa5-l, ebruary 2
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Subsequently, the pGEM1 construct was digested with EcoRI-HincII and Co.) was internalized for the indicated times at 37(cid:56)C. After fixation, Tf 23
ligated into the Mx expression cassette EcoRI-SnaBI. was visualized using a sheep anti–human Tf serum followed by FITC-
labeled anti–sheep IgG and Rab17 with a polyclonal rabbit antibody fol-
Generation and Characterization of Stable Inducible lowed by rhodamine-labeled donkey anti–rabbit secondary antibody.
Eph4 Cell Lines
Eph4 cells were cotransfected in suspension with constructs Mx-rab17 Colocalization of Rab17 and Internalized Fab Fragment
WT, Mx-rab17N132I, or Mx-rab17Q77L and SV40neo for selection using in Eph4 Cells
DOTAP (Boehringer Mannheim). Colonies were selected and maintained
in 800 (cid:109)g/ml G418 (geneticin; GIBCO BRL). Expressing colonies were Fab fragment was prepared as outlined above (preparation and biotinyla-
screened by indirect immunofluorescence after induction with interferon tion of the Fab fragment). Eph4 cells harboring Mx-rab17 WT were plated
(Sigma Chemical Co.). Positive clones were expanded, plated on filter on coverslips and treated with interferon to induce the expression of both
supports (Costar Corp.), and transepithelial resistance was measured. rab17 and the FcLR 5-27. Fab fragment was added to the cells in a volume
Only those lines with a transepithelial resistance above 2,000 ohm/cm2 of 200 (cid:109)l at a concentration of 7 (cid:109)g/ml for 5, 10, 20, 40, and 60 min at 37(cid:56)C.
were used for subsequent studies. Zo-1 was also visualized to ensure that Cells were then rinsed in PBS, fixed, and processed for indirect immuno-
the lines were polarized. The lines were subcloned to obtain a high num- fluorescence. The Fab fragment was visualized using an affinity-purified
ber of expressing cells. goat anti–rat IgG F(ab(cid:57)) fragment specific antibody followed by
2
A second round of transfection was carried out to introduce Mx-FcLR rhodamine-labeled donkey anti–goat secondary antibody. Rab17 was vi-
5-27 into the above cell lines. Cotransfection was carried out as above us- sualized using a polyclonal rabbit antibody followed by FITC-labeled don-
ing cells in suspension and SV40-puro (Clontech Laboratories Inc., Palo key anti–rabbit IgG. Samples were analyzed by confocal microscopy.
Zacchi et al. Rab17 and Polarized Transport 1041
Confocal Microscopy, Data Acquisition, and Analysis with NHS-SS-biotin (Pierce) as previously described (Smythe et al., 1992).
Eph4 cells grown either on plastic or on filter supports were serum starved
Samples were visualized using the EMBL confocal scanning laser beam 8 h in DME containing 10 mM Hepes and 0.2% BSA in the presence or
microscope. Confocal series were carried out at either 0.5 or 1 (cid:109)m steps. absence of interferon. For single round of transferrin cycle experiments,
The 529 and 476 laser lines of the Argon-ion laser were used for excitation cells were first incubated with 125 nM of biotinylated transferrin in inter-
of rhodamine/propidium iodide and fluorescein, respectively. In the case nalization medium (DME, 10 mM Hepes, 0.5% BSA) for 1 h on ice (sur-
of Fig. 5, the data were collected on a Leica confocal TCS-NT microscope. face binding). The biotinylated transferrin was added to the basolateral
Images were recorded and imported into either Adobe Photoshop or NIH side in the case of filter grown Eph4 cells. After washing six times with
Image graphic programs for compilation, and printed directly using the cold PBS(cid:49)/0.5% BSA cells were incubated with internalization medium
Kodak Color Ease printer. for various times at 37(cid:56)C. The medium of plastic-grown Eph4 cells or the
apical and basolateral media of filter-grown Eph4 cells was collected and
Endocytosis, Transcytosis, and Recycling of the FcLR the surface-bound biotinylated transferrin was stripped by reduction with
5-27 Chimeric Receptor MesNa (Matter et al., 1993). The intracellular signal associated with the
filters was determined by lysing the cells as described above. For continu-
Endocytosis and transcytosis mediated by chimeric receptors were as- ous uptake experiments, biotinylated transferrin was added to the basolat-
sayed as previously described (Hunziker and Mellman, 1989). Briefly, Fab eral medium of filter-grown Eph4 cells for 90 min at 37(cid:56)C. The apical me-
fragments derived from monoclonal antibody 2.4G2 (Unkeless, 1979) dium was collected, the cells were extensively washed, and then lysed. The
were biotinylated with NHS-LC-biotin (Pierce, Rockford, IL) as de- ECL counts of the lysates, and collected media were determined as de-
scribed above (preparation and biotinylation of Fab fragment section). scribed below.
Saturating concentrations of biotinylated-Fab fragments (0.5–10 (cid:109)g/ml) The leakage of the cells was controlled in each experiment both with
were prebound apically or basolaterally to filter grown Eph4 cells for 1 h the transferrin receptor and the FcLR 5-27 receptor. In experiments
on ice. Unbound antibody fragments were removed by washing five times where the ligand was prebound on either side of the monolayer on ice, the
with PBS(cid:49)/0.5% BSA and twice with PBS(cid:49). To determine the apical– excess ligand was washed away and the filters incubated at 37(cid:56)C for vari-
basolateral surface distribution of the FcLR 5-27 chimeric receptor, the ous time intervals. We checked the initial leakage after prebinding by Do
cell surface–associated Fab fragments were eluted with medium contain- stripping the ligand from both cell surfaces at time zero after washing wn
ing 0.5% BSA, pH 3.4. To measure intracellular transport, after binding, away excess ligand in duplicate filters. After prebinding and washing away load
the cells were incubated for different lengths of time in MEM without excess ligand, the filters were incubated at 37(cid:56)C for various lengths of ed
phenol red (Sigma Chemical Co.) containing 10 mM Hepes, pH 7.4, and time. For each time point at 37(cid:56)C, duplicate filters were incubated on ice. from
subsequently cooled down on ice. Apically and basolaterally bound anti- The leakage was controlled by stripping the ligand from both cell surfaces h
bmoind,i ees luwaetrees ewluetreed cwoiltlehc mteedd aiunmd ceolunttiaoinn iwnga s0 .r5e%pe BatSeAd ,o pnHce 3. .T4.h Ae fpteHr 1o0f awth tehree stahme eco tnimtine uaosu tsh ue p3t7a(cid:56)kCe soafm trpalnessf wererrine fporro c9e0s mseidn. aItn 3t7h(cid:56)eC e wxpaes rqimuaennttis- ttp://rup
Tthhees ei netlruaacteelsl uwlaars saidgjnuaslt eads stooc 7ia.0te bdy w aidtdhi nthge 4 f0i–lt6e0r s(cid:109) wl oafs 1d eMte Hrmepineesd, pbHy 7ly.6s-. flaieyde,r .duplicate filters were incubated on ice to check leakage of the mono- ress.o
mdimneiMgsn cte rhMsiecbeg ecnCdec lle2bls) e (iclEnoo wCnKt.L OaU)inA pcinocta guBk n1eut% sfvf eoa Trflu r(tei1htso1e 5n ree mXlpurM-ae1ts0 eeK0sn Oaat nnAtddhc e0,l y.a25sm5%a tmoe uSsM nDwt S Heo.r efeE p sldeiegescn,tt eaprrolH mcr hei7nes.i4ems,dt ia2 luan.5s-t EThlee cEtrCoLc-Ahneamlyisleur mSyinsteemsc ewnasc ep uDrcehtaesecdt iofrnom S yIgsetne mInc. (Rockville, rg/jcb/article-p
to acid elution from both surfaces. Transcytosis is calculated as the per- MD). Affinity-purified goat anti–rat IgG F(ab(cid:57))2 fragment specific anti- df/1
centage of signal in the corresponding acid eluate relative to the total body and rabbit anti–sheep IgG were labeled with Origen Tag-NHS 40
amount of originally bound Fab fragment. (N-hydroxysuccinimide ester of a ruthenium chelate; Igen, Inc.) following /5/1
0
To measure recycling of the chimeric receptor to the apical plasma the protocol described by the manufacturer (Igen, Inc.). The ECL counts 39
membrane, saturating concentration of Fab fragments reversibly deriva- of the surface-bound (acid-releasable) and intracellular (acid-resistant) /12
tized with biotin (see above) were first preinternalized apically for 10 min at biotinylated Fab for the transcytotic assay were determined as follow: 50 79
5
37(cid:56)C. The medium was then removed and the cells were further incubated (cid:109)l of the eluates and lysates were incubated with 4 (cid:109)g/ml ruthenium- 95
for 10 min at 37(cid:56)C in the presence of 100-fold excess of nonbiotinylated labeled affinity-purified goat anti–rat IgG F(ab(cid:57))2 fragment specific anti- /29
3
Fab. Cells were then cooled on ice, washed five times with PBS(cid:49)/0.5% body for 2 h at RT. Thereafter, 1 (cid:109)l of M-280 Streptavidin Dynabeads 98
BSA, and biotin bound to Fab fragments remaining at the cells surface (Dynal, Hamburg, Germany) was added for 15 min at RT. The reaction .pd
fw5o0ar s m2 s0Mt rmi pMipnee fsdoN lblaoy,w 1ree0dd0 u bmcyt Mitoh nrNe wea iCwthla, sM2h.e5es sm NwMait h( tC PharBCeSel(cid:49)2 ,w c5ao0sn hmteasMi niin nT gfrr i2es0-sHh mlCyM lp, rpieoHpda o7rae.c8d-, wdiarWse cstittloyhp qrpeuesadpn ebtciyft i aetddod btirnya gtn h2sef0e 0Er r(cid:109)CinlL ot far anafsafsliayckyze ibnru.gf, fae rd (iIfgfeerne nInt cp.)r oatnodc osal mwapsle sse wt eurpe. f by guest o
edtiaummi dfoer; Mvaartitoeurs etti male.,s 1a9t9 337).(cid:56) CT.h Be iocetilnlsy lwaeterde -Finacbu bfraategdm einn tcso, mrepclyectlee dm teo- T1 h(cid:109)el osfa mMp-2le8s0 cstornetpatianviindgin b Dioytninaybleaatdeds, tthraenns wfeirthri n1. 2w5e (cid:109)rel oinf cshuebeapte adn tfii–rhstu wmiatnh n 22 F
the apical membrane or transcytozed to the basolateral surface, were transferrin serum (SAPU, Law Hospital Carluke, Scotland) diluted in 400 eb
eluted and the ECL counts of the eluates and of the lysates were deter- (cid:109)l of wash buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2% Triton rua
mined as described below. X-100, 0.2% BSA), and then finally with 2 (cid:109)g/ml ruthenium-labeled rab- ry 2
0
bit anti–sheep IgG. All the incubation steps were performed at RT for 1 h. 23
Nocodazole Treatment Between the different incubation steps, M-280 streptavidin Dynabeads
were washed three times with wash buffer using a Dynal MPC-E Magnetic
Nocodazole (Sigma Chemical Co.) was dissolved in DMSO at 33 mM Particle Concentrator (Dynal, Oslo, Norway). After the last washing step
stock solution in DMSO, stored at (cid:50)20(cid:56)C. Polarized Eph4 cells were ei- the Dynabeads were resuspended in 100 (cid:109)l of wash buffer and 100 (cid:109)l of
ther pretreated 3 h at 37(cid:56)C or 60 min at 4(cid:56)C after allowing the internaliza- assay buffer and subsequently quantified by the ECL-analyzer. Both as-
tion from the basolateral side of biotinylated Fab fragments for 30 min at says were validated by comparing the results to those obtained using iodi-
37(cid:56)C in the presence of 33 (cid:109)m nocodazole. The apical and basolateral an- nated Fab fragment and iodinated transferrin.
tibody fragments were eluted and quantified. In control experiments,
Eph4 cells were incubated in the presence of DMSO alone. Nocodazole
Results
did not increase the paracellular diffusion of the Fab fragments in either
direction, suggesting that the drug did not affect the integrity of the cell
monolayer. Immunofluorescence was also performed to visualize the ex- Targeting of Rab17 in Nonpolarized Cells
tent of microtubule depolymerization under the experimental conditions
used. Samples were fixed with paraformaldehyde using the pH shift proto- In polarized epithelial cells of the mouse kidney tubules,
col (Bomsel et al., 1989).
rab17 was found mainly associated with the basolateral
plasma membrane and apical dense tubules (Lütcke et al.,
Analysis of Transferrin Transport
1993). To determine whether rab17 localizes to endocytic
Iron-saturated human transferrin (Sigma Chemical Co.) was biotinylated organelles, we first studied the targeting of the protein ec-
The Journal of Cell Biology, Volume 140, 1998 1042
topically expressed in nonpolarized BHK-21 cells using IM-2 cell line (Reichmann, 1989) and selecting for pure
the vaccinia VT7 system (Bucci et al., 1992). By immuno- epithelial single cell colonies. The cells resemble mam-
fluorescence confocal microscopy, the bulk of the labeling mary gland luminal epithelium, and can be stimulated un-
appeared as a strong focus of fluorescence concentrated in der certain conditions to produce (cid:98)-casein (Strange et al.,
the perinuclear area of the cell and in discrete vesicles dis- 1991). Although to date there is almost no data regarding
persed throughout the cytoplasm (Fig. 1, A, D, and G). their trafficking properties, we used Eph4 cells instead of
This staining pattern was reminiscent of the pericentriolar the most currently used MDCK cell line for several rea-
recycling compartment in which Tf and TR accumulate at sons: (a) it responds to mouse interferon; (b) both rab17
steady state (Ghosh et al., 1994; Hopkins et al., 1994). We, cDNA and the cell line are derived from the same species
therefore, investigated whether rab17 positive structures (Lütcke et al., 1993), thus avoiding potential problems of
were accessible to internalized Tf. WT rab17 and the hu- mistargeting in nonrodent cell lines (Lütcke, A., and M.
man transferrin receptor (hTR) were coexpressed in Zerial, unpublished observations); (c) it contains endoge-
BHK-21 cells and FITC-Tf was internalized for different nous rab17 (Fig. 2, B and C); (d) the line is nontumori-
lengths of time. As shown in Fig. 1 B, after 15 min inter- genic and, based on the expression of epithelial marker
nalization at 37(cid:56)C, FITC-Tf localized in peripherally dis- proteins and the high transepithelial resistance, it exhibits
tributed punctate structures, consistent with a localization a polarized phenotype (Reichmann et al., 1989, 1992). We
to early endosomes. At this point, some colocalization generated stable Eph4 cell lines which overexpress WT
with rab17 could be detected (Fig. 1, A–C). After 30 and rab17 and two mutants defective in either GTP-hydrolysis
60 min internalization, however, FITC-Tf concentrated in (rab17Q77L) or nucleotide binding (rab17N132I). An in-
D
the pericentriolar area of the cell where it now colocalized ducible expression system was used because constitutive o
w
with the bulk of rab17 (Fig. 1, D–F and G–I). Thus, when expression of dominant interfering mutants of other rab nlo
a
ectopically expressed in nonpolarized cells, rab17 is prima- proteins (Bucci et al., 1994) has been reported to be toxic de
d
rily targeted to the perinuclear recycling endosome. to mammalian cells. For this purpose, the well character- fro
m
ized interferon (IFN)- inducible Mx promoter (Hug et al., h
Inducible Expression of Rab17 in Polarized Eph4 1988; Arnheiter et al., 1990) was chosen because of its tight ttp://ru
inducibility (Bachiller and Rüther, 1990; Arnheiter et al., p
Epithelial Cells re
1990). The rab17 open reading frame was cloned under ss.o
Traob 1st7u diny tphoel alroiczaeldiz aetpiiotnh ealniadl tcheell fsu, nwceti otonoalk r oadlev panlatyaegde boyf (cid:98)th-eg lcoobnintr ionlt roofn t hwisa sp urosemdo ttoe rm, aasx iimlluizsetr tartaends cinri pFtiigo.n 2 ( AW. oTohde- rg/jcb/a
the Eph4 cell line. Eph4 cells originate from the mammary roofe et al., 1992). Furthermore, we engineered a consensus rticle
gland epithelium, by sucloning the previously described Kozak site in all constructs to enhance translation efficiency -pd
f/1
4
0
/5
/1
0
3
9
/1
2
7
9
5
9
5
/2
9
3
9
8
.p
d
f b
y g
u
e
st o
n
2
2
F
e
b
ru
a
ry 2
0
2
3
Figure 1. Colocalization of rab17 WT and in-
ternalized FITC-labeled transferrin in nonpo-
larized cells. BHK-21 cells overexpressing
rab17 WT and human transferrin receptor by
vaccinia infection and transfection (see Mate-
rials and Methods) were incubated for 15
(A–C), 30 (D–F), and 60 min (G–I) in the
presence of FITC-labeled transferrin. Cells
were then fixed, permeabilized, and rab17
was visualized using a polyclonal rabbit anti-
body followed by rhodamine-labeled donkey
anti–rabbit IgG. Samples were analyzed by
confocal microscopy. A, D, and G show rab17
staining pattern, B, E, and H show FITC-
labeled transferrin distribution, and C, F, and
I represent the overlays. Bar, 10 (cid:109)m.
Zacchi et al. Rab17 and Polarized Transport 1043
Figure 2. Generation of Eph4 cell lines and characterization of
D
the interferon-inducible system. (A) Construct used to generate ow
n
the Eph4 stable lines overexpressing rab17 WT, rab17Q77L, and lo
a
rab17N132I. The amino acid changes are indicated in shadow de
d
text. (B) Time course of protein expression after induction by in- fro
m
terferon: protein expression from the Mx promoter was induced h
with 500 U of interferon. Protein extracts were prepared 0, 3.5, ttp
10, 24, and 48 h after induction, blotted, and probed with a poly- ://ru
p
cinlotenrafle arontni bwoadsy wreacsohgendi zoiungt aranbd1 c7e. lFlsi nwaellrye, ainftceurb iantdeudc tfioorn a f ofur r2t4h ehr, ress.o
rg
24 h in the absence of interferon. (C) Concentration of interferon /jcb
rweaqsu iinreddu cfeodr wmitahx iinmcurema sipnrgo tueninit s eoxfp irnetsesrifoenr:o np rforotemin 0 etox p1r,e0s0s0io Un /article
per ml. Protein extracts were prepared 24 h after induction, blot- -pd
ted, and probed with a polyclonal antibody recognizing rab17. f/14
0
/5
/1
0
3
9
/1
2
7
(Kozak, 1987). Time and dose dependence of expression 9
5
9
were assayed by Western blot analysis. Treatment of Eph4 5/2
Figure 3. Localization of rab17 9
cells grown on plastic support for 24 h with increasing 39
WT protein in polarized Eph4 8
amounts of IFN led to a concentration-dependent expres- .p
sion, reaching a plateau at 500 U (Fig. 2 C). To estimate cfieltllesr. sEupphp4o rctes,l lrsa wb1e7r eW gTro wwans ionn- df by g
twhieth t i5m00e Uco oufr sIeF No ff oerx pvraersiosiuosn l,e nEgpthh4s ocfe ltlism we.e Prer oitnedinu ceexd- dwuecreed fwixiethd inatnedr feprroonc,e assnedd ceflolsr uest on
pression was detected already at the earliest time point in- confocal analysis. Cells were 22
F
vestigated (3.5 h) and was maximal after 10 h of induction fixed, permeabilized, and rab17 was visualized using a polyclonal eb
(Fig. 2 B). Moreover, by confocal immunofluorescence mi- rabbit antibody followed by FITC-labeled donkey anti–rabbit rua
croscopy analysis performed on Eph4 cell lines grown on IgG (A, D, G, J, M, and P). Nuclei were visualized with propid- ry 2
0
filter support, rab17 was confirmed to be homogeneously ium iodide (B, E, H, K, N, and Q). C, F, I, L, O, and R represent 23
the overlays. Samples were analyzed by serial sectioning from the
expressed in the cells (not shown). For all the experiments
apical cell surface (ACS; A–C) and the following sections are
reported here, Eph4 cells were incubated for 8 h in the
shown: 1.6 (cid:109)m below the ACS (D–F), 2.4 (cid:109)m below the ACS (G–I),
presence of 500 U of IFN. 3.2 (cid:109)m below the ACS (J–L), 5.6 (cid:109)m below the ACS (M–O), and
8.4 (cid:109)m below the ACS (P–R). Bar, 5 (cid:109)m.
Intracellular Distribution of Rab17 in Polarized
Eph4 Cells
The subcellular localization of rab17 was then examined in of the cell above the nucleus and underneath the apical
filter-grown Eph4 cells harboring Mx-rab17 WT by immu- plasma membrane, as previously shown on mouse kidney
nofluorescence confocal microscopy. Serial sections were sections (Lütcke et al., 1993). These structures extended
recorded from the apical region of the cell just below the also towards the lateral side, but the signal was undetect-
apical brush border (Fig. 3, A–C), above (Fig. 3, D–F) and able on the basolateral plasma membrane. The lack of la-
at the level of the nucleus (Fig. 3, G–I), from the lateral beling on the basolateral plasma membrane is in contrast
surface of the cell (Fig. 3 J–O) and from the basal portion with what we previously observed in kidney cells (Lütcke
of the cell (Fig. 3, P–R). Rab17 appeared to be concen- et al., 1993). The reasons for this discrepancy are not clear
trated in vesicular and tubular elements at the apical pole and could be due to antibody reactivity or differences in
The Journal of Cell Biology, Volume 140, 1998 1044
D
o
w
n
lo
a
Figure 4. Immunoelectron microscopic localization of rab17 in de
d
Eph4 cells. Cryosections of Eph4 cells were labeled with antibod- Figure 5. Colocalization of rab17 WT and internalized holo- from
iaetse dto w riatbh1 t7u fboullloowveesdic buyla 1r0 e nlemm pernotste (iAn –AC-)g,o alds .w Lealbl ealsi nwgi tish amssuolctii-- transferrin in Eph4 cells grown on plastic. Holo Tf was internal- http
vesicular endosomes (e, land A; also see inset in B). Labeled ized for 5 (A), 15 (B), 30 (C), and 60 min (D) at 37(cid:56)C in Eph4 ://ru
ttuhbe uplloavsemsaic muleamr berleamnee n(ptsm w) aenred cfrloesqeu teon tcleyn torbiosleersv e(cd, luanned eBrl)y. iAngs cseetll sin h aAr bsohroinwg aMt hx-igrahb m17a WgnTif iacaftteior nin sdturucctitounre ws iwthh ienrtee rrfaebr1o7n .a Innd- press.o
ssbhouomdwse,nt ia mlitneh soA sue,g ehtnh ettho er ba cbeo 1ia7nt- epcdoo nsritteiingvuieoi tntysu wbwuietlhore vp euisnticavutailrvaiear bcellyale tmhrarebinn1–t7sc- onwaetegerade- talarlaibzneesldefe dru rasiinnnt gic– osalho escheapelie zIpeg G(aan.r tRrio–awhbus1)m7. aCwnea lsTl svf w isseuerareuli mzfiex def odul slaoinnwdge aTd fp bwoyla ysFc lIvoTinsCua--l rg/jcb/article
tive. Double labeling with antibodies to cellubrevin followed by rabbit antibody followed by rhodamine-labeled donkey anti–rab- -pd
5 nm protein A–gold (inset, B) showed some colocalization of cel- bit secondary antibody. Bars, 20 (cid:109)m; inset, 10 (cid:109)m. f/14
0
lubrevin (small gold, arrowheads) and rab17 (large gold). n, nu- /5/1
cleus. Bars, 100 nm. 03
9
/1
molecule would enter the rab17-positive compartment in 2
7
9
Eph4 cells. In the first part of our analysis we used semi- 59
5
steady state distribution in various cell types. Most impor- polarized cells grown on coverslips to gain maximal sensi- /29
3
tantly, the staining pattern in the apical region is strikingly tivity of detection of the fluorescent markers. The distribution 98
.p
sniomrimlaarl ltyo lothcaatt eodf athroeu anpdi ctahle r ceecnyctrlionsgo mened (oAsopmodea, cwah eict ha li.s, oinfg h WumTa rna bh1o7l ofo-Tr fv ainrtioeurns atliimzeeds aint 3E7p(cid:56)Ch 4w caesl lcso omvpearreexdp rweistsh- df by g
u
1ra9b9147; Mimomstuonvo ealnedct rCoanr dmoincreo, s1co99p5y) .a nAallsyosi,s ionn tEhep hc4a scee llosf, tmhiactr oofs croapby1.7 A byfe dr o5u mblien- luapbteal kceo,n Tfof cwaal sim pmreusennotf liuno preusnccetnactee est on
2
harboring Mx-rab17 WT grown on plastic showed the structures dispersed throughout the cell periphery, whereas 2 F
e
presence of the protein on tubular structures, which sur- the bulk of rab17 was concentrated in the perinuclear bru
a
round the centrosome (Fig. 4 B), on vesicular elements lo- compartment with some label also in small peripheral ves- ry 2
cated close to the plasma membrane (Fig. 4, A and C), and icles radiating from it. Only few of these structures also 02
3
on multivesicular endosomal structures (Fig. 4, A and inset contained Tf (Fig. 5 inset A), suggesting that rab17 is not
B). These latter structures were positive for the synapto- enriched in basolateral early endosomes. After 15 min in-
brevin/VAMP–related protein cellubrevin (McMahon et ternalization, only a small fraction of Tf reached the rab17
al., 1993; Galli et al., 1994; Daro et al., 1996), a membrane in the pericentriolar recycling endosome. Interestingly, lit-
protein ubiquitously distributed along the receptor-medi- tle colocalization at this time point was especially observed
ated endocytic pathway, confirming the endosomal nature in more confluent and polarized cells where the rab17 posi-
of rab17-labeled elements. tive compartment was positioned above the nucleus (Fig. 5
B). However, the colocalization increased after 30 and 60
min, when Tf progressively entered the rab17-positive
Transferrin Internalized from the Basolateral
strucures located on the apical pole of the cells (Fig. 5, C
Cell Surface Colocalizes with Rab17 in an Apical
and D).
Endosomal Compartment
To examine the accessibility of the rab17 positive com-
Because it has been reported that at least a fraction of TR, partment to Tf in fully polarized Eph4 cells, the human TR
internalized from the basolateral surface into basolateral was transiently overexpressed using a recombinant herpes
early endosomes, accesses the apical recycling compart- virus (Geller and Breakefield, 1988), as the endogenous
ment (Apodaca et al., 1994), we initially assayed whether this labeling was too low to be detected in filter-grown cells
Zacchi et al. Rab17 and Polarized Transport 1045
Figure 6. Colocalization of rab17 and internalized FITC-labeled D
o
transferrin in polarized epithelial cells. Eph4 cells harboring wn
lo
Mx-rab17 WT were grown on filter supports, induced with in- a
d
e
terferon to express the rab17 protein and FITC-labeled trans- d
ferrin was internalized from the basolateral surface for 90 min from
at 37(cid:56)C. Cells were washed, fixed, and rab17 was visualized us- http
ing a polyclonal rabbit antibody followed by rhodamine-labeled ://ru
donkey anti–rabbit IgG. Samples were analyzed by confocal mi- pre
croscopy. A, D, G, and J show FITC transferrin; B, E, H, and K ss.o
show rab17; and C, F, I, and L represent the overlays. Samples rg
were serially sectioned from the ACS and as shown in the dia- /jcb
/a
gJ–raLm, 2 t o(cid:109) mth eb erilgohwt ,t hAe– AC CarSe. B0.a5r (cid:109), 1m0, (cid:109)Dm–F. 1 (cid:109)m, G–I, 1.5 (cid:109)m, and rticle-p
d
f/1
4
0
/5
Effect of Rab17 Mutants on Transferrin Recycling /10
(data not shown). The coding region of the hTR was 39
/1
cloned into pHSV-1 amplicon vector, and was subsequently Given that internalized Tf is able to reach rab17-positive 2
7
9
packaged using a replication-incompetent HSV-1 helper endosomal structures both in nonpolarized and polarized 59
5
virus in a permissive cell line (Lim et al., 1996). The virus cells, we analyzed whether the overexpression of WT and /29
3
produced was used to infect Eph4 cells harboring Mx- mutant rab17 proteins would affect the Tf cycle. Because 98
.p
rab17 WT construct (Murphy et al., 1997). To compare the Eph4 cells display low levels of TR, we used a highly sensi- df b
distribution of rab17 with the intracellular compartments tive nonradioactive detection system based on ECL tech- y g
u
tehraatll yT Rin htearsn aacliczeesds tfoo ra t9 s0t emadiny satta t3e7,(cid:56) FCI.T CCo-Tnffo wcaals sbeacstoiolants- ncyoclloingyg twoi tqhuoauntt itfhye t hnee ekdin teot iocvse orfe Txpf rienstse rTnRal i(zsaeteio Mn aatnedr iraels- est on
2
scanning specifically the apical region of the cells, each 0.5 and Methods). The Tf cycle was initially studied in plastic- 2 F
(cid:109)m apart, are shown in Fig. 6. A significant degree of colo- grown Eph4 cells treated with or without IFN to compare ebru
a
cnaalliizzaetdio Tnf bweatsw oebesne rrvaebd1 7th srtoauingihnogu at nthde b aapsioclaalt emroalslty r eingtieorn- tBhieo tkininyelatitcesd i nT ft hwea sp rfiersset nbcoeu onrd aobns etnhcee s uorf fraacbe1 a7t p4r(cid:56)Cot,e ainnsd. ry 202
3
of the cells (Fig. 6, C, F, I, and L, arrows). In addition, then internalized by shifting the cells to 37(cid:56)C. Fig. 7 shows
some structures labeled for rab17 could be observed that the kinetics of the Eph4 clone overexpressing the rab17N132I
were poorly labeled with FITC-Tf, indicating that rab17 mutant protein (Fig. 7, A and B). In all cell lines tested,
resides on apical structures that are not accessible to, or prebound transferrin was rapidly endocytosed and effi-
enriched in FITC-Tf endocytosed from the basolateral ciently recycled back to the plasma membrane. The mea-
side. These results indicate that rab17-positive apical struc- sured rates are similar before and after induction of the
tures, at least partially, overlap with the apical endosomal rab17 proteins, indicating that the Tf cycle is not affected
compartment accessible by Tf internalized from the baso- by the overexpression of WT and mutants rab17 in nonpo-
lateral membrane. It has been speculated that the apical larized Eph4 cells.
recycling endosome may correspond to the recycling en- We then performed similar experiments in polarized
dosome described in nonpolarized cells (Apodaca et al., rab17N132I Eph4 cell lines grown on filter supports. Tf en-
1994). Given that rab17, when ectopically expressed in docytosis and basolateral recycling were similarly unaf-
nonpolarized cells, localizes to perinuclear recycling endo- fected (Fig. 7, C and D). In contrast, we observed a small
somes, these results provide support for the hypothesis but significant increase of apically released Tf in cells ex-
that recycling endosome and apical recycling compartment pressing rab17Q77L and rab17N132I mutants (data not
are homologous organelles. shown), which was neither seen in the case of WT rab17
The Journal of Cell Biology, Volume 140, 1998 1046
Figure 7. Transferrin internalization and re-
cycling in Eph4 cells. Eph4 cells were plated
on plastic dishes: when 80% confluent, half of
the wells were induced with interferon.
D
Transferrin internalization and recycling in o
w
Eph4 cells harboring Mx-rab17N132I (A and nlo
a
B) were measured as outlined in Materials de
and Methods, minus ((cid:104)) and plus ((cid:100)) inter- d fro
feron induction. In C and D Eph4 cells har- m
h
boring Mx-rab17N132I were grown on filter ttp
supports, half of the filters were induced with ://ru
p
interferon and transferrin was prebound to re
the basolateral surface. Transferrin recycled ss.o
rg
tuoe st,h me inbuass o(l(cid:104)at)e aranld mpleudsi u((cid:100)m) ainntde rifnetreornn ainl dvuacl-- /jcb/a
tion, were measured. In E Parental Eph4 cells rticle
and Eph4 cells harboring Mx-rab17 WT, Mx- -pd
rab17Q77L, and Mx-rab17N132I were grown f/14
0
on filter supports and half of the filters were induced with interferon. Transferrin was continuously internalized from the basolateral sur- /5
face for 90 min at 37(cid:56)C. Then the cells were rapidly cooled on ice, the apical media collected, and measured as previously described (see /103
9
Materials and Methods). The given values represent the mean of duplicate samples expressed relative to the same cell line without inter- /1
2
feron induction; the experiments were repeated three times with similar results. 79
5
9
5
/2
9
3
9
8
.p
d
FcLDL Receptor Chimera Has Access to f b
protein overexpression, nor was it due to the IFN induc- Rab17-positive Endosomal Compartment y gu
tioTno i tbseeltft e(rd afotall onwot tshheo twranf)f.icking of transferrin to the api- The observation that a fraction of TR is transcytosed api- est on
2
cal cell surface, we changed the experimental conditions in cally in response to the expression of rab17 mutants, 2 F
e
order to follow multiple transferrin cycles. Tf was continu- prompted us to investigate more closely the transcytotic bru
a
oatu s3l7y(cid:56) Cin.t eArnt athliizse dti mfreo mpo tihnet, bthaseo flraatectriaoln s uorff atrcaen fsofre r9r0in m rien- pthaeth FwcaLyR i n5 -t2h7e s(eM caetltlesr. Teto a tlh., i1s 9e9n3d),, aw ech tiomoekr iacd rveacnetpatgoer oinf ry 202
3
leased by Eph4 cells into the apical medium in the absence which the extracellular and the transmembrane domain of
or presence of IFN was (cid:122)19% of the total cell associated the mouse IgG Fc receptor have been fused to the cyto-
(surface-bound and internal). The amount recovered in plasmic tail of the LDL receptor deleted of the distal baso-
the apical medium of the various Eph4 cell lines was ex- lateral sorting determinant (Matter et al., 1993). When ex-
pressed as a percentage of the same cell line before induc- pressed in MDCK cells, this receptor is targeted to both
tion with IFN. As shown in Fig. 7 E, we detected a 50% in- apical and basolateral plasma membrane domains. In this
crease of apically released Tf in cells overexpressing rab17 way it is possible to determine apical and basolateral up-
mutants compared with uninduced or control cells, indi- take and transcytosis occurring in both directions (apical
cating that interfering with the function of rab17 causes an to basolateral and basolateral to apical; Matter et al., 1992,
increase of Tf release into the apical medium. The effect 1993). We measured the receptor traffic using a well char-
on Tf transcytosis was observed only after induction of the acterized high affinity Fab fragment of the anti-FcRII
rab17 mutant proteins. The lines harboring the mutant monoclonal antibody 2.4G2 as a pseudoligand (Unkeless,
proteins had a similar receptor number to the Eph4 paren- 1979; Mellman and Plutner, 1984a; Mellman et al., 1984b).
tal line or the line expressing rab17 WT, ruling out the pos- We intended to constitutively express the FcLR 5-27 in
sibility that the increased apically released Tf may be due the above described rab17 Eph4 cell lines. However, all at-
to increased receptor number. tempts were unsuccessful, and we, therefore, inducibly ex-
Zacchi et al. Rab17 and Polarized Transport 1047
vesicles, some colocalizing with rab17 (Fig. 8, A–C), but
after 10 min a small fraction of the Fab fragment started to
enter the rab17-positive compartment in the perinuclear
area of the cell (Fig. 8, D–F). The colocalization of inter-
nalized Fab fragment and rab17 significantly increased af-
ter 20 min of internalization (Fig. 8, G–I) and was maximal
at 40 and 60 min at 37(cid:56)C (Fig. 8, J–L and M–O). These re-
sults show that FcLR 5–27 does access the rab17-positive
compartment, indicating that it is a suitable marker to fur-
ther investigate the transport steps regulated by rab17.
Rab17 Mutants Affect Transcytosis of the
FcLDL 5-27 Receptor
We next analyzed the effect of rab17 mutants on FcLR 5–27
trafficking along both apical and basolateral endocytic
pathways. As in the case of transferrin measurements, we
made use of a nonradioactive detection system to deter-
mine the amount of Fab fragment surface bound, endocy-
D
tosed and transcytosed by the Eph4 clones. Biotinylated o
w
Fab fragments of the anti-FcRII antibody were prebound nlo
a
either at the apical or at the basolateral surface of the de
d
Eph4 cells. After extensive washing, cells were incubated fro
for different times at 37(cid:56)C and surface-bound antibody m h
fragments were eluted from both plasma membrane do- ttp://ru
mains by low pH treatment. The remaining Fab fragment p
re
therefore represents the fraction of internalized receptor. ss.o
Ionn tthhee Esuprhf4a ccel ownaes c1o9n%tro al ptihcea ld aisntdri b8u1t%io nb aosfo tlhaete rreacl.e Tpthoirs rg/jcb/a
was similar to the distribution in Eph4 cells expressing rticle
rab17WT (25% apical and 75% basolateral). However, in -pd
rab17Q77L and rab17N132I expressing cell lines more re- f/14
0
ceptor was found on the apical surface (32% apical, 68% /5/1
0
basolateral and 42% apical, 58% basolateral, respec- 39
/1
tively). Thus, the mutant proteins altered the apical–baso- 2
7
9
lateral distribution of the FcLR 5-27. 59
5
In all Eph4 clones expressing rab17Q77L and rab17N132I /29
3
Figure 8. Colocalization of rab17 and internalized Fab fragment. examined, the chimeric receptor was efficiently internal- 98.p
Fab fragment was internalized for 5 (A–C), 10 (D–F), 20 (G–I), ized at both apical and basolateral surfaces, as presented df b
40 (J–L), or 60 min (M–O) at 37(cid:56)C in Eph4 cells harboring Mx- in Fig. 9 A. The measured rate of uptake from the apical y g
u
rwaibth1 7i nWteTrf earnodn M. Cxe-Fllcs LwRe r5e– f2i7x erdec aenpdto trh ceo Fnastbr ufrcatsg mafetenrt iwndasu cvtiisoun- awnitdh tthheo sbea soofl attheer aEl pshu4rf acceell wlineree eexspsreenstsiianlgly thcoem rpecaerapbtoler est on
2
baFle(ialzeebdd(cid:57) ) 2db oyf nriankgdemyir eeancnttt iism–pgmeocauitfn icos eflacuonontridbeaosrcdyey n acfneot,li lbuooswidneygd. aRb gyao bar1th7 oa nwdtaaim–s rivanites Iu glaaGl-- ainlogn We (Tc lroanbe1 c7o (ndtarotal) naos tw sehlol wasn t)h, es uEgpghe4st icnegll tlhinaet enxopnree sosf- 2 Februa
ized using a polyclonal rabbit antibody followed by FITC-labeled the rab17 proteins affected these steps. In contrast, we ry 2
donkey anti–rabbit IgG. Samples were analyzed by confocal mi- found that rab17Q77L and rab17N132I mutants caused a 023
(cid:122)2.5-fold increase in basolateral to apical transcytosis of
croscopy. A, D, G, J, and M show rab17 staining pattern, B, E, H,
K, and N represent the Fab staining pattern and C, F, I, L, and O the receptor (Fig. 9 B). This effect was neither seen in the
represent the overlays. Bars, 20 (cid:109)m. Eph4 clone control nor in the cells expressing WT rab17
(not shown), and was detectable only after 20 min or
longer incubation time. It is important to note that, be-
pressed the receptor using the Mx promoter. Eph4 cell cause the results are expressed as a percentage of Fab
lines harboring Mx-rab17 constructs were subsequently fragment initially bound and because the counts of baso-
stably transfected with the Mx-FcLR 5-27 construct and lateral binding and internalization are higher than the cor-
selected for inducible expression of the protein. responding ones for the apical side, the magnitude of the
We first verified that, as predicted, the FcLR 5-27 would basolateral to apical transcytosis is underestimated com-
reach the rab17 positive compartment. The intracellular pared with that for the opposite direction (Fig. 9 B). This
localization of the FcLR 5-27 was assayed in plastic-grown is not the case, as in Eph4 parental cells and the line ex-
Eph4 cells overexpressing WT rab17 by continuous uptake pressing WT rab17, the flow of transport in both the baso-
of the Fab fragment for different lengths of time. After 5 lateral to apical and the apical to basolateral transcytosis
min of internalization at 37(cid:56)C the Fab fragment labeling was quantitatively similar (see legend to Fig. 9). The
was dispersed throughout the cytoplasm in small discrete rab17Q77L overexpressing cell line showed just a moder-
The Journal of Cell Biology, Volume 140, 1998 1048