Basolateral Sorting of the Cation-dependent Mannose 6-Phosphate Receptor in Madin-Darby Canine Kidney Cells
1998; Elsevier BV; Volume: 273; Issue: 1 Linguagem: Inglês
10.1074/jbc.273.1.186
ISSN1083-351X
AutoresBen Distel, Ulrike Bauer, Roland Le Borgne, Bernard Hoflack,
Tópico(s)Barrier Structure and Function Studies
ResumoIn polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1. In polarized Madin-Darby canine kidney (MDCK) cells, sorting of membrane proteins in the trans-Golgi network for basolateral delivery depends on the presence of cytoplasmic determinants that are related or unrelated to clathrin-coated pit localization signals. Whether these signals mediate basolateral protein sorting through common or distinct pathways is unknown. The cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains clathrin-coated pit localization signals that are necessary for endocytosis and lysosomal enzyme targeting. In this study, we have addressed the function of these signals in polarized sorting of the CD-MPR. A chimeric protein, made of the luminal domain of the influenza virus hemagglutinin fused to the transmembrane and cytoplasmic domains of the CD-MPR was stably expressed in MDCK cells. This chimera (HCD) is able to interact with the AP-1 Golgi-specific assembly proteins and is detected on the basolateral plasma membrane of MDCK cells where it is endocytosed. Deletion analysis and site-directed mutagenesis of the cytoplasmic domain of the CD-MPR indicate that HCD chimeras devoid of clathrin-coated pit localization signals are still transported to the basolateral membrane where they accumulate. A HCD chimera containing only the transmembrane domain and the 12 membrane-proximal amino acids of the CD-MPR cytoplasmic tail is also found on the basolateral membrane but is unable to interact with the AP-1 assembly proteins. However, the overexpression of this mutant results in partial apical delivery. It is concluded, therefore, that the basolateral transport of this chimera requires a saturable sorting machinery distinct from AP-1. The plasma membrane of polarized cells can be divided into two distinct domains, apical and basolateral, which exhibit different protein and lipid compositions. The generation and the maintenance of these domains require a continuous supply of newly synthesized components. In MDCK 1The abbreviations used are: MDCK, Madin-Darby canine kidney; TGN, trans-Golgi network; MPR, mannose 6-phosphate receptor; CD-MPR, cation-dependent mannose 6-phosphate receptor; HA, hemagglutinin; IGF, insulin-like growth factor; PAGE, polyacrylamide gel electrophoresis; Man-6-P, mannose 6-phosphate; MEM, minimum Eagle's medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin. cells, newly synthesized membrane proteins destined for the basolateral or the apical surface are sorted in the trans-Golgi network (TGN) and packaged into distinct transport vesicles (1Wandering-Ness A. Bennett M.K. Antony C. Simons K. J. Cell Biol. 1990; 111: 987-1000Crossref PubMed Scopus (216) Google Scholar). Vesicular transport from the TGN to the apical or basolateral plasma membrane domains are mechanistically different. Although the docking/fusion of transport vesicles with the basolateral plasma membrane relies, like many transport steps, on the presence of the common fusion machinery involving NSF/SNAP proteins, the apical delivery appears to be independent of these proteins (2Ikonen E. Tagaya M. Ullrich O. Montecucco C. Simons K. Cell. 1995; 81: 571-580Abstract Full Text PDF PubMed Scopus (222) Google Scholar). More recent studies have indicated that nonpolarized cells also make use of two types of transport intermediates for the delivery of membrane proteins to the plasma membrane, one dependent on the presence of NSF/SNAP proteins and another independent of these complexes (3Yoshimori T. Keller P. Roth M.G. Simons K. J. Cell Biol. 1996; 133: 247-256Crossref PubMed Scopus (202) Google Scholar). Thus, polarized and nonpolarized cells have fairly similar overall organization of membrane traffic within the secretory pathway. Thus far, two distinct features have been shown to determine sorting to the apical domain: first, the glycosylphosphatidylinositol anchor of membrane proteins (4Brown D.A. Crise B. Rose K. Science. 1989; 245: 1499-1501Crossref PubMed Scopus (304) Google Scholar, 5Lisanti M.P. Caras I.W. Davitz M.A. Rodriguez-Boulan E. J. Cell Biol. 1989; 109: 2145-2156Crossref PubMed Scopus (375) Google Scholar) and second, the mannose-rich core part ofN-glycans present in the luminal domain of proteins (6Scheiffele P. Peränen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar). Many studies have now illustrated that sorting of membrane proteins to the basolateral plasma membrane is determined by the presence of specific, dominant protein determinants in their cytoplasmic domains (reviewed in Ref. 7Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (393) Google Scholar). Extensive mutagenesis has uncovered two types of sorting motifs for basolateral delivery. First, there are those related to signals for clathrin-coated pit localization, which either rely on a key tyrosine residue, like those found in the LDL receptor (proximal determinant) (8Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar), the vesicular stomatatis virus G protein (9Thomas D.N.C. Brewer C.B. Roth M.G. J. Biol. Chem. 1993; 268: 3313-3320Abstract Full Text PDF PubMed Google Scholar), and lysosomal membrane glycoproteins (10Höning S. Hunziker W. J. Cell Biol. 1995; 128: 321-332Crossref PubMed Scopus (117) Google Scholar), or on a di-leucine motif, like in the IgG Fc receptor (11Hunziker W. Fumey C. EMBO J. 1994; 13: 2963-2969Crossref PubMed Scopus (220) Google Scholar). Second, there are basolateral targeting signals that are unrelated to determinants for clathrin-coated pit localization. Examples can be found in the LDL receptor (distal determinant) and in the poly-IgA receptor (8Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 12Casanova J.E. Apodaca G. Mostov K. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 13Aroeti B. Kosen P.A. Kuntz I.D. Cohen F.E. Mostov K.E. J. Cell Biol. 1993; 123: 1149-1160Crossref PubMed Scopus (118) Google Scholar). Interestingly, the same (or a very closely related) basolateral sorting signal can mediate the recycling of endocytosed membrane proteins from endosomes back to the plasma membrane (14Aroeti B. Mostov K. EMBO J. 1994; 13: 2297-2304Crossref PubMed Scopus (89) Google Scholar, 15Matter K. Whitney J.A. Yamamoto E.M. Mellman I. Cell. 1993; 74: 1053-1064Abstract Full Text PDF PubMed Scopus (133) Google Scholar). The similarities between the determinants responsible for endocytosis, basolateral sorting, and plasma membrane recycling suggest that these processes are extremely related and involve similar sorting machineries that remain to be characterized. In addition to sorting membrane proteins destined for the apical or basolateral domains in the TGN, the polarized MDCK cells must also sort their newly synthesized lysosomal hydrolases bound to the mannose 6-phosphate receptors (MPRs). Previous studies have shown that one of the two MPRs, the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II), traffics within the basolateral domain of MDCK cells because it can be detected on the basolateral membrane of these cells (16Prydz K. Brändli A.W. Bomsel M. Simons K. J. Biol. Chem. 1990; 265: 12629-12635Abstract Full Text PDF PubMed Google Scholar). In nonpolarized cells, the endocytosis of this receptor relies on a tyrosine-based motif, whereas that of the other MPR, the cation-dependent mannose 6-phosphate receptor (CD-MPR), requires a weak tyrosine-based motif and a dominant motif containing two phenylalanine residues (17Johnson K.F. Chan W. Kornfeld S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 10010-10014Crossref PubMed Scopus (88) Google Scholar). On the other hand, efficient lysosomal enzyme targeting requires the presence of a di-leucine-based motif present at the carboxyl terminus of both MPRs (18Johnson K.F. Kornfeld S. J. Biol. Chem. 1992; 267: 17110-17115Abstract Full Text PDF PubMed Google Scholar, 19Johnson K.F. Kornfeld S. J. Cell Biol. 1992; 119: 249-257Crossref PubMed Scopus (175) Google Scholar, 20Chen H.J. Remmler J. Delaney J.C. Messner D.J. Lobel P. J. Biol. Chem. 1993; 268: 22338-22346Abstract Full Text PDF PubMed Google Scholar). In addition, the signals required for efficient endocytosis of the MPRs contribute, although weakly, to efficient lysosomal enzyme targeting (19Johnson K.F. Kornfeld S. J. Cell Biol. 1992; 119: 249-257Crossref PubMed Scopus (175) Google Scholar). The MPRs and their bound lysosomal enzymes are known to be sorted in the TGN via clathrin-coated vesicles. The first step in the formation of these transport intermediates is the interaction of the AP-1 Golgi-specific assembly proteins with TGN membranes. The MPRs are part of the membrane components that permit the efficient recruitment of AP-1 on membranes (21Le Borgne R. Schmidt A. Mauxion F. Griffiths G. Hoflack B. J. Biol. Chem. 1993; 268: 22552-22556Abstract Full Text PDF PubMed Google Scholar, 22Le Borgne R. Griffiths G. Hoflack B. J. Biol. Chem. 1996; 271: 2162-2170Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 23Le Borgne R. Hoflack B. J. Cell Biol. 1997; 137: 335-345Crossref PubMed Scopus (88) Google Scholar), a process regulated by the small GTPase ARF-1 (24Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar). In the case of the CD-MPR, specific determinants in its cytoplasmic domain, in particular a casein kinase II phosphorylation site are required for high affinity interaction of AP-1 with TGN membranes (25Mauxion F. Le Borgne R. Munier-Lehmann H. Hoflack B. J. Biol. Chem. 1996; 271: 2171-2178Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In this study, we have investigated the sorting of the CD-MPR in polarized MDCK cells. For this, we have stably expressed a chimeric protein made of the luminal domain of the influenza virus hemagglutinin (HA) fused to the transmembrane and cytoplasmic tail of the CD-MPR. This HCD chimeric protein traffics within the basolateral domain and can be detected at the basolateral surface. Mutations of the different sorting signals proposed to mediate the interaction of the CD-MPR tail either with the Golgi-specific assembly proteins AP-1 or its plasma membrane counterpart AP-2 do not affect the basolateral delivery of the corresponding HCD chimeras. Truncations of the cytoplasmic domain indicated that a sorting determinant, unrelated to motifs necessary for clathrin-coated pit localization, is present in the membrane-proximal part of the CD-MPR cytoplasmic domain or the transmembrane domain, which confers basolateral targeting. This determinant, neither supports the AP-2-dependent endocytosis nor triggers the recruitment of AP-1 on membranes. This strongly suggests that an additional sorting machinery that recognizes signals unrelated to those mediating clathrin-coated pit localization could be responsible for basolateral targeting of membrane proteins in MDCK cells. MDCK cells (strain II) were grown as described (26Matlin K.S. Reggio H. Helenius A. Simons K. J. Cell Biol. 1981; 91: 601-613Crossref PubMed Scopus (465) Google Scholar). PA317 amphotropic retrovirus packaging cells (27Miller A.D. Buttimore C. Mol. Cell. Biol. 1989; 6: 2895-2901Crossref Scopus (1145) Google Scholar) were maintained in MEM supplemented with 10% fetal calf serum, 4 mmglutamine, and antibiotics. All experiments, unless otherwise indicated, were performed with MDCK cells grown on 24.5-mm diameter, 0.4-mm pore size transwell units (Costar, Cambridge, MA). The polycarbonate filters were seeded with 0.5 to 1 × 106cells each, and cell monolayers were used for experiments 4 days after plating. All manipulations of DNA were performed essentially as described in Ref. 28Sambrook J Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar. To optimize expression of the chimeric proteins a Kozak consensus sequence was introduced into the cDNA encoding HA. Two complementary oligonucleotides with the sequences 5′-CAAGCTTGCCGCCACCATGG-3′ and 5′-CCATGGTGGCGGCAAGCTTGGTAC-3′ were ligated between the KpnI and MscI sites of plasmid pBHA (29Mauxion F. Schmidt A. Le Borgne R. Hoflack B. Eur. J. Cell Biol. 1995; 66: 119-126PubMed Google Scholar) to create pBD16. To form pBD17 (wtHA), pBD16 was cleaved with HindIII and BamHI and the short fragment encoding the complete influenza virus hemagglutinin protein was cloned between EcoRI and BamHI sites of the retroviral vector pLXSN (30Miller A.D. Rosman G.J. BioTechniques. 1989; 7: 980-990PubMed Google Scholar) after making the HindIII and EcoRI ends flush with Klenow. The construction of a chimeric gene, HCD, consisting of the luminal domain of the influenza virus HA fused to the transmembrane and cytoplasmic domains of the small MPR has been described previously (29Mauxion F. Schmidt A. Le Borgne R. Hoflack B. Eur. J. Cell Biol. 1995; 66: 119-126PubMed Google Scholar). The chimeric gene HCD and all mutants derived thereof were inserted in pBD17 between the SalI and BamHI sites, thereby fusing the 5′ end of HA, containing the Kozak consensus sequence, to all chimeric genes. The cytoplasmic domain truncation mutants (HCD-Δ5 (pBD23), HCD-Δ17 (pBD20), and HCD-Δ55 (pBD26); see Fig. 1) and the substitution mutants (HCD-Δ55,A-1 (pBD28) and HCD-A13A18A45 (pBD19)) were generated by oligonucleotide-directed mutagenesis of the wild type mouse CD-MPR cDNA (31Ludwig T. Ruther U. Metzger R. Copeland N.G. Jenkins N.A. Lobel P. Hoflack B. J. Biol. Chem. 1992; 267: 12211-12219Abstract Full Text PDF PubMed Google Scholar) using the method of Kunkel (32Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). For the truncation mutants the codons for the CD-MPR cytoplasmic tail residues 63 (His) and 51 (Gln) were changed to stop codons. In the case of the substitution mutants, the last residue (Tyr) of the transmembrane domain and the cytoplasmic tail residues Phe13, Phe18, and Tyr45 were changed to alanines. The truncation mutant HCD-Δ23 (pBD25) was generated by ligation of two complementary oligonucleotides with the sequences 5′-GCATGATTGG-3′ and 5′-GATCCCAATCAT-3′ between the PstI and BamHI sites of HCD, introducing a stop codon at Tyr45 in the cytoplasmic tail of the CD-MPR. Mutant HCD-Δ55 (pBD26) and Δ55,A-1 (pBD30) were constructed in a similar way using oligonucleotides with the sequences 5′-ATGGAGCAGTGAG-3′ and 5′-GATCCTCACTGCTCCATTCCC-3′. This double-stranded DNA fragment was inserted between the BstXI and BamHI sites of HCD and HCD-A-1, respectively, creating a stop codon at residue 13 (Phe) of the cytoplasmic domain. Infectious virus was generated for each of the constructs from the plasmid form of the retroviral vector by transient transfection of the packaging cells PA317 essentially as described (30Miller A.D. Rosman G.J. BioTechniques. 1989; 7: 980-990PubMed Google Scholar). Virus-containing medium harvested from PA317 cells was used to infect MDCK cells. Stably transfected MDCK cells were selected in medium containing 0.8 mg/ml G418 (Life Technologies, Inc.) and cloned using glass cylinders. Cells expressing the protein of interest were identified by immunofluorescence. At least two independent clones expressing the highest levels of the respective chimeric protein were used for further experiments. All transfected cell lines used were fully polarized as judged by methionine uptake (basal/apical ratio greater than 4:1) (33Balcarova-Stander J. Pfeiffer S.E. Fuller S.D. Simons K. EMBO J. 1984; 3: 2687-2694Crossref PubMed Scopus (144) Google Scholar) and secretion of an endogenous glycoprotein complex (34Urban J. Parczyk K. Leutz A. Kayne M. Kondor-Koch C. J. Cell Biol. 1987; 105: 2735-2743Crossref PubMed Scopus (146) Google Scholar). Prior to experiments the tightness of monolayers was assessed with [3H]inulin (Amersham Corp.) (35Brewer C.B. Roth M.G. J. Cell Biol. 1991; 114: 413-421Crossref PubMed Scopus (211) Google Scholar). All experiments were performed using cell lines of passage numbers 5 through 10 after cloning. MDCK cells grown on Costar Transwell units were rinsed with warm PBS++ (PBS containing 0.9 mm CaCl2 and 0.5 mmMgCl2) and starved for 60 min in MEM lacking cysteine and methionine (Select Amine-kit; Life Technologies, Inc.) containing 0.35 g/liter sodium bicarbonate, 20 mm Hepes, pH 7.3, and 0.5% BSA (MEM-BSA). Cells were then pulse labeled in a wet chamber for 20 min at 37 °C from the basolateral side with labeling medium (MEM-BSA supplemented with 2 mCi/ml Expre35S35S (1000 Ci/mmol, 10 mCi/ml) NEN Life Science Products). One set of filters was washed three times with cold PBS++ and placed on ice in MEM-BSA; the other sets of filters were chased at 37 °C in MEM-BSA with a 100-fold excess methionine and cysteine. At the end of the chase cells were washed with cold PBS++ and placed on ice in MEM-BSA. Subsequent steps were performed at 4 °C. Cells were washed once with MEM-BSA and incubated either from the apical or basolateral side with a 1:500 dilution of an hemagglutinin antiserum (monoclonal antibody H269, generous gift of J. Skehel) in MEM-BSA for 90 min on a rocking platform. In some experiments the antibody was included in the apical or basolateral chase medium and allowed to bind for another 90 min on ice. The excess unbound antibodies were removed over 30 min by three washes with MEM-BSA and one wash with PBS++, filters were cut out of the holder, and cells were lysed in the presence of an excess of unlabeled protein. (For preparation of unlabeled lysates a cell line overexpressing the wild type HA was grown to confluency on 10-cm culture dishes. Cells from one dish were lysed on ice in 2.5 ml of B1 (50 mm Tris, pH 7.2, 100 mm NaCl, 2 mm EDTA, 1% Triton X-100, 0.1% SDS containing freshly added protease inhibitor mixture (2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride)). The lysate was cleared by centrifugation for 5 min at 12,000 × g, and 1 ml of supernatant was used to lyse labeled cells.) The lysate was centrifuged in an Eppendorf microfuge for 5 min to remove debris. An aliquot (one-tenth of the lysate) was supplemented with additional antibody (anti-HA, mAb H269) and incubated overnight at 4 °C, and total labeled protein was isolated by the addition of protein A-Sepharose. The second aliquot (nine-tenths of the lysate) was frozen in liquid nitrogen and stored overnight at −70 °C. Labeled protein that had appeared on the cell surface and thus had bound antibody was precipitated by the addition of protein A-Sepharose. Precipitates were washed three times with B1, twice with B2 (50 mm Tris, pH 7.2, 100 mm NaCl, 2 mm EDTA, 0.1% Triton X-100, 0.5% SDS, 0.5% deoxycholate), twice with B3 (50 mm Tris, pH 7.2, 500 mm NaCl, 2 mm EDTA, 0.1% Triton X-100), and once with B4 (50 mm Tris, pH 7.2, 100 mm NaCl, 2 mm EDTA). Finally, proteins were released from the beads by boiling in Laemmli sample buffer and analyzed by SDS-PAGE on a 10% polyacrylamide gel (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar). The band intensities were calculated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and the amount of protein transported to the cell surface was expressed as the percentage of the total immunoprecipitated protein. In some experiments cells were pretreated with 10 mm butyric acid (Sigma) 12 h prior to labeling to induce transcription of stably transfected cDNA constructs (8Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar). MDCK cells grown on 60-mm plastic dishes to subconfluency and treated with 10 mm NH4Cl 12–16 h prior to the experiment to reduce the endogenous level of lysosomal proteases were metabolically labeled essentially as described above except that cells were labeled for 1 h and chased for 3 h. After labeling cells were lysed, and an aliquot of the lysate was analyzed by sucrose gradient centrifugation as described (37Copeland C.S. Doms R.W. Bolzau E.M. Webster R.G. Helenius A. J. Cell Biol. 1986; 103: 1179-1191Crossref PubMed Scopus (246) Google Scholar). Gradient fractions were immunoprecipitated with anti-HA and analyzed by SDS-PAGE and fluorography. Internalization rates of CD-MPR chimeras were determined by the surface biotinylation assay described in Ref. 38Prill V. Lehmann L. Von Figura K. Peters C. EMBO J. 1993; 12: 2181-2193Crossref PubMed Scopus (87) Google Scholar, except that MESNa was used for stripping of the cell surface biotin. Biotinylated CD-MPR chimera were detected by Western blotting with125I-labeled streptavidin. Signals were quantified using a PhosphorImager. HeLa cells were grown on coverslips in α-MEM supplemented with 10% fetal calf serum, 10 mmHepes, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were washed in medium devoid of serum and then incubated for 30 min with a recombinant virus that expresses the T7 polymerase gene (39Fuerst T.R. Niles E.G. Studier F.W. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8122-8126Crossref PubMed Scopus (1874) Google Scholar). After washing the cells in medium supplemented with 5 mm hydroxyurea, theN-[1(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium-methylsulfate reagent (Boehringer Mannheim) was used to transfect the cells with the indicated constructs (HCD and HCD-Δ55) cloned in pGEM1 vector, following the manufacturer's instructions. After 1 h, the cells were washed and allowed to express for 2–3 h in medium supplemented with 5 mm hydroxyurea. Pulse-chase experiments performed in parallel indicated that similar levels of HCD and HCD-Δ55 were expressed under those conditions and transported through the secretory pathway with similar kinetics (data not shown). Cells were then fixed for 20 min with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100 for 5 min, and subsequently incubated with the monoclonal 100/3 anti-γ-adaptin antibody (kindly provided by E. Ungewickell) and a rabbit polyclonal anti-HA antibody for 30 min at room temperature. The bound antibodies were detected with fluorescein isothiocyanate or Texas Red-conjugated secondary antibodies (Dianova). For quantitation of the fluorescent signal, randomly chosen fields were captured using a cooled charged and coupled device CH250 from Photometrics (Tuckson, AZ) having a Kodak KAF 1400 CCD CHIP (grade 2) for 12-Bit image collection that was controlled by the KHOROS software package running on a SUN 10/41 workstation (Silicon Graphics, USA). In every field, regions corresponding to the TGN area of transfected or nontransfected cells were selected, and the fluorescence in the Texas Red channel corresponding to the 100/3 monoclonal antibody was calculated using the AIM program developed by J. C. Olivo from the EMBL microcomputing and data acquisition group. After calculating the fluorescence intensities (mean intensity/pixel or total intensity) from 88 (HCD transfected), 122 (nontransfected), and 73 (HCD-Δ55) cells, mean and standard error were calculated. The confidence limits of the results obtained were assessed by Student's t test. In MDCK cells, the Man-6-P/IGF II receptor traffics between the TGN and the endocytic organelles of the basolateral domain and is found on the basolateral plasma membrane (16Prydz K. Brändli A.W. Bomsel M. Simons K. J. Biol. Chem. 1990; 265: 12629-12635Abstract Full Text PDF PubMed Google Scholar). To study the transport of the CD-MPR in these cells, we have generated MDCK clones stably expressing chimeric proteins made of the luminal domain of the influenza virus HA, an apically sorted membrane glycoprotein, fused to the transmembrane and cytoplasmic domain of this receptor (Fig.1) and performed pulse-chase experiments followed by cell surface immunoprecipitations to measure the appearance of these chimeric proteins on the apical or the basolateral domain. The normal transport of HA to the cell surface depends on its proper folding and trimerization (37Copeland C.S. Doms R.W. Bolzau E.M. Webster R.G. Helenius A. J. Cell Biol. 1986; 103: 1179-1191Crossref PubMed Scopus (246) Google Scholar). Because modifications in the transmembrane and cytoplasmic domains of HA can affect its normal rate of transport (40Doyle C. Roth M.G. Sambrook J. Gething M.-J. J. Cell Biol. 1985; 100: 704-714Crossref PubMed Scopus (65) Google Scholar, 41Doyle C. Sambrook J. Gething M.-J. J. Cell Biol. 1986; 103: 1193-1204Crossref PubMed Scopus (57) Google Scholar) we determined whether the HA/CD-MPR chimera (HCD construct) is properly folded and transported through the secretory pathway. We first performed classical pulse-chase experiments on MDCK cells expressing either the wild type HA or the HCD and immunoprecipitated these labeled proteins using a monoclonal antibody directed against the luminal domain of HA. After a 20-min pulse, the HA and the HCD were found in low molecular weight, immature forms that were rapidly converted upon a chase into higher molecular weight, mature forms reflecting the conversion of high mannose to complex type oligosaccharides (Fig. 2). These results indicate that the HCD chimera moves efficiently through the secretory pathway with similar rates as the HA (t½ ≈ 30 and 25 min, respectively). Such pulse-chase experiments were also performed with the HCD-Δ55, which harbors a 55-amino acid-long truncation of the CD-MPR tail (Fig. 2). The rate of transport of this mutant was slightly slower than that of HCD (t½ ≈ 45 min), indicating that the deletion of the CD-MPR tail only moderately affects the transport of the corresponding chimera. The other mutants used in this study were transported through the secretory pathway with similar rates as that of HCD-Δ55 (data not shown). The oligomeric state of HCD and HCD-Δ55 were analyzed by centrifugation of detergent extracts on sucrose density gradients (37Copeland C.S. Doms R.W. Bolzau E.M. Webster R.G. Helenius A. J. Cell Biol. 1986; 103: 1179-1191Crossref PubMed Scopus (246) Google Scholar). After the pulse, HCD and HCD-Δ55 immunoprecipitated from the different fractions distributed in a single peak, as the monomeric HA (Fig.3). After a chase period, however, both chimeric proteins with complex type sugars were recovered in denser fractions, showing a similar profile as the mature trimeric HA. A slightly higher percentage of the HCD-Δ55 remains in the lighter density fractions in comparison with HA or HCD following a 3-h chase. This suggests that the rate of oligomerization of the trucation mutant is slightly slower than that of HA or HCD. Nevertheless, these results show that both chimeric proteins acquire the same trimeric conformation as HA.Figure 3Oligomerization of wild type HA and HA/CD-chimeras. Transfected cells were either pulse labeled with [35S]methionine/cysteine for 1 h (P) or pulse labeled for 1 h followed by a 3-h chase (C). Cell lysates were centrifuged on 5–25% (w/v) sucrose gradients containing 0.1% Triton X-100. Each fraction was immunoprecipitated with anti-HA and analyzed by SDS-PAGE and fluorography.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next performed cell surface immunoprecipitations on biosynthetically labeled MDCK cells grown on filters to monitor the appearance of the newly synthesized wild type HA and HCD on the apical or the basolateral surface (Fig. 4). As found previously (35Brewer C.B. Roth M.G. J. Cell Biol. 1991; 114: 413-421Crossref PubMed Scopus (211) Google Scholar, 42Matlin K.S. Simons K. J. Cell Biol. 1984; 99: 2131-2139Crossref PubMed Scopus (130) Google Scholar) the bulk of the wild type HA was transported to and accumulated at the apical plasma membrane. Most of the newly synthesized HCD, however, progressively appeared on the basolateral plasma membrane. Typically, 90% of the cell surface HCD was found on the basolateral plasma
Referência(s)