Retrograde Transport of KDEL-bearing B-fragment of Shiga Toxin
1997; Elsevier BV; Volume: 272; Issue: 31 Linguagem: Inglês
10.1074/jbc.272.31.19554
ISSN1083-351X
AutoresLudger Johannes, Danièle Tenza, Claude Antony, Bruno Goud,
Tópico(s)Clostridium difficile and Clostridium perfringens research
ResumoTo investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation. To investigate retrograde transport along the biosynthetic/secretory pathway, we have constructed a recombinant Shiga toxin B-fragment carrying an N-glycosylation site and a KDEL retrieval motif at its carboxyl terminus (B-Glyc-KDEL). After incubation with HeLa cells, B-Glyc-KDEL was progressively glycosylated in the endoplasmic reticulum (ER) and remained stably associated with this compartment. B-fragment with a nonfunctional KDEL sequence (B-Glyc-KDELGL) was glycosylated with about the same kinetics as B-Glyc-KDEL but localized at steady state to the Golgi apparatus. Morphological studies showed that B-Glyc-KDEL was delivered from the plasma membrane, via endosomes and the cisternae of the Golgi apparatus, to the ER. Moreover, the addition of a sulfation site allowed us to show that B-Glyc-KDEL on transit to the ER entered the Golgi apparatus through the trans-Golgi network. Transport of B-Glyc-KDEL to the ER was slowed down by nocodazole, indicating that microtubules are important for the retrograde pathway. Our results document the existence of a continuous pathway from the plasma membrane to the endoplasmic reticulum via the Golgi apparatus and show that a fully folded exogenous protein arriving in the endoplasmic reticulum via this pathway can undergo N-glycosylation. The existence of retrograde transport along the biosynthetic/secretory pathway was first demonstrated for a certain class of luminal resident ER 1The abbreviations used are: ER, endoplasmic reticulum; TGN, trans-Golgi network; Lamp, lysosomal-associated protein; PBS, phosphate-buffered saline; DMM, 1-deoxymannojirimicin; DTAF, 5([4,6-dichlorotriazin-2-yl]amino)fluorescein. proteins carrying the carboxyl-terminal tetrapeptide KDEL (in mammals) or HDEL (in yeast). Both biochemical and morphological evidence indicates that these proteins can leave the ER, reach the Golgi apparatus in which they acquire Golgi-specific carbohydrate modifications, and be subsequently retrieved to the ER (1Dean N. Pelham H.R. J. Cell Biol. 1990; 111: 369-377Crossref PubMed Scopus (126) Google Scholar, 2Hsu V.W. Yuan L.C. Nuchtern J.G. Lippincott-Schwartz J. Hammerling G.J. Klausner R.D. Nature. 1991; 352: 441-444Crossref PubMed Scopus (130) Google Scholar, 3Jackson M.R. Nilsson T. Peterson P.A. J. Cell Biol. 1993; 121: 317-333Crossref PubMed Scopus (314) Google Scholar, 4Lewis M.J. Pelham H.R. Cell. 1992; 68: 353-364Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 5Pelham H.R. Hardwick K.G. Lewis M.J. EMBO J. 1988; 7: 1757-1762Crossref PubMed Scopus (189) Google Scholar). The retrieval of ER-escaped proteins could occur from locations as distal as thetrans-Golgi network (TGN) (6Miesenbock G. Rothman J.E. J. Cell Biol. 1995; 129: 309-319Crossref PubMed Scopus (112) Google Scholar). ER resident membrane proteins can also leave the ER and are retrieved from the Golgi apparatus. This process involves basic residues located at the carboxyl- or amino-terminal ends of the proteins (7Jackson M.R. Nilsson T. Peterson P.A. EMBO J. 1990; 9: 3153-3162Crossref PubMed Scopus (727) Google Scholar, 8Schutze M.P. Peterson P.A. Jackson M.R. EMBO J. 1994; 13: 1696-1705Crossref PubMed Scopus (268) Google Scholar). Golgi resident proteins may also undergo retrograde movement. For instance, the medial Golgi protein MG160 is sialylated, suggesting that it cycles between late Golgi/TGN compartments in which sialylation occurs and medial Golgi (9Johnston P.A. Stieber A. Gonatas N.K. J. Cell Sci. 1994; 107: 529-537PubMed Google Scholar). Retrograde movement has recently been documented for other yeast and mammalian Golgi proteins, such ascis-Golgi mannosyltransferase Och1p, medial/trans-GolgiN-acetylglucosaminyltransferase I, and Golgi-localized Emp47p (10Harris S.L. Waters M.G. J. Cell Biol. 1996; 132: 985-998Crossref PubMed Scopus (123) Google Scholar, 11Hoe M.H. Slusarewicz P. Misteli T. Watson R. Warren G. J. Biol. Chem. 1995; 270: 25057-25063Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Schroder S. Schimmoller F. Singer-Kruger B. Riezman H. J. Cell Biol. 1995; 131: 895-912Crossref PubMed Scopus (157) Google Scholar). Other evidence for the existence of a retrograde pathway comes from work on some bacterial toxins that seem to enter the cytosol of higher eukaryotic cells after reaching ER compartments (13Pelham H.R.B. Roberts L.M. Lord J.M. Trends Cell Biol. 1992; 2: 183-185Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Particularly well studied examples are Shiga toxin from Shigella dysenteriaeand Shiga-like toxins (or verotoxins) from Escherichia coli. These toxins are composed of two polypeptidic chains, one of which (A-fragment) carries a deadenylase activity that inhibits protein biosynthesis by acting on the 28 S rRNA, whereas the other subunit (B-fragment) allows the binding of the toxin to target cells (14O'Brien A.D. Tesh V.L. Donohue-Rolfe A. Jackson M.P. Olsnes S. Sandvig K. Lindberg A.A. Keusch G.T. Curr. Top. Microbiol. Immunol. 1992; 180: 65-94Crossref PubMed Scopus (328) Google Scholar). Electron microscopic analysis has shown that Shiga toxin can be detected in the ER of T47D cells, of butyric acid-treated A431 cells, and of Daudi cells (15Garred O. Dubinina E. Holm P.K. Olsnes S. van Deurs B. Kozlov J.V. Sandvig K. Exp. Cell Res. 1995; 218: 39-49Crossref PubMed Scopus (60) Google Scholar, 16Khine A.A. Lingwood C.A. J. Cell. Physiol. 1994; 161: 319-332Crossref PubMed Scopus (69) Google Scholar, 17Sandvig K. Garred O. Prydz K. Kozlov J.V. Hansen S.H. van Deurs B. Nature. 1992; 358: 510-512Crossref PubMed Scopus (378) Google Scholar, 18Sandvig K. Ryd M. Garred O. Schweda E. Holm P.K. van Deurs B. J. Cell Biol. 1994; 126: 53-64Crossref PubMed Scopus (153) Google Scholar). Shiga toxin does not carry a KDEL motif at its carboxyl terminus, and the mechanism of its retrograde transport is currently unknown. In contrast, retrieval signals are found on cholera toxin and Pseudomonas exotoxin A (19Chaudhary V.K. Jinno Y. FitzGerald D. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 308-312Crossref PubMed Scopus (217) Google Scholar, 20Majoul I.V. Bastiaens P.I. Soling H.D. J. Cell Biol. 1996; 133: 777-789Crossref PubMed Scopus (137) Google Scholar). For ricine it has been shown that the addition of a KDEL peptide to the toxin increases its toxicity (21Wales R. Chaddock J.A. Roberts L.M. Lord J.M. Exp. Cell Res. 1992; 203: 1-4Crossref PubMed Scopus (54) Google Scholar). The aim of this study was to investigate further the retrograde transport pathway. For this purpose, we constructed a recombinant B-fragment that was carboxyl-terminally modified by the addition of anN-glycosylation site and the KDEL peptide (B-Glyc-KDEL). We found that a significant part of this chimeric protein was glycosylated after incubation with HeLa cells, indicating that it can be transported to the ER. The presence of the KDEL motif was not critical for retrograde transport per se, but for retention of B-Glyc-KDEL in the ER. A biochemical and morphological analysis allowed us to show that B-Glyc-KDEL passed in a microtubule-dependent fashion from endosomes via the TGN/Golgi apparatus to the ER. HeLa cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.), 0.01% penicillin/streptomycin, 4 mm glutamine, and 5 mm pyruvate in a 5% CO2 incubator. Mouse hybridoma cells expressing the monoclonal anti-VT1 antibody 13C4 were purchased from American Type Culture Collection (CRL 1794) and kept in culture according to ATCC instructions. Monoclonal antibody 13C4 was purified from culture medium on protein A-Sepharose (Pharmacia Biotech Inc.). The monoclonal anti-Lamp-2 antibody H4B4 was purchased from PharMingen (San Diego); the monoclonal anti-transferrin receptor antibody H68.4 and the polyclonal antibodies anti-signal sequence receptor and anti-galactosyltransferase were kindly provided by I. Trowbridge (The Salk Institute, San Diego), T. A. Rapoport (Harvard University, Boston), and E. Berger (Institute of Physiology, University of Zurich, Switzerland), respectively. To construct a plasmid expressing B-Glyc-KDEL and B-Glyc-KDELGL, a two-step polymerase chain reaction-based strategy was adopted essentially as described previously (22Johannes L. Lledo P.M. Roa M. Vincent J.D. Henry J.P. Darchen F. EMBO J. 1994; 13: 2029-2037Crossref PubMed Scopus (187) Google Scholar). Polymerase chain reaction primers ShigaC-4 (for B-Glyc-KDEL; 5′-ACTAGCTCTGAAAAGGATGAACTTTGAGAATTCTGACTCAGAATAGCTC-3′) or ShigaC-5 (for B-Glyc-KDELGL; 5′-ACTAGCTCTGAAAAGGATGAACTTGGTCTTTGAGAATTCTGACTCAGAATAGCTC-3′) and ShigaC-3 (5′-CTTTTCAGAGCTAGTAGAATTAGGATGATAGCGGCCGCTACGAAAAATAACTTCGC-3′) were used with plasmid pSU108 (a generous gift from K. N. Timmis, Gesellschaft für biotechnologische Forschung, Braunschweig, Germany; (23Su G.F. Brahmbhatt H.N. Wehland J. Rohde M. Timmis K.N. Infect. Immun. 1992; 60: 3345-3359Crossref PubMed Google Scholar)) specific primers ShigaAtpE (5′-CACTACTACGTTTTAAC-3′) and Shiga-fd (5′-CGGCGCAACTATCGG-3′) to produce fragments that were cloned into the SphI andSalI restriction sites of pSU108. Sequences derived by polymerase chain reaction were verified by dideoxy sequencing (Pharmacia). A pSU108-based plasmid expressing B-Glyc-Sulf-KDEL was constructed essentially as described previously (24Niehrs C. Huttner W.B. Ruther U. J. Biol. Chem. 1992; 267: 15938-15942Abstract Full Text PDF PubMed Google Scholar). Briefly, sulfation sites encoding 5′-phosphorylated oligonucleotides Sulfat5 (5′-GAGGAACCTGAGTATGGAGAA-3′) and Sulfat6 (5′-CCTTTCTCCATACTCAGGTTC-3′) were hybridized and ligated at 16 °C for 8 h. Adaptor fragments containing the N-glycosylation site and the KDEL sequence, composed of oligonucleotides Sulfat1 (5′-phosphorylated; 5′-GGCCGCCATCCTAATTCTACTTCT-3′) and Sulfat2 (5′-CTCAGAAGTAGAATTAGGATGGC-3′), or of Sulfat3 (5′-GAGTCTGAAAAAGATGAACTTTGATGAG-3′) and Sulfat4 (5′-phosphorylated; 5′-AATTCTCATCAAAGTTCATCTTTTTCAGA-3′) were ligated overnight at 16 °C. The resulting fragment was cloned into the NotI and EcoRI restriction sites of pSU108 containing the cDNA coding for B-Glyc-KDEL (see above). Oligonuclotide-derived sequences were verified by dideoxy sequencing (Pharmacia). Purification of recombinant B-fragments was essentially done as described (23Su G.F. Brahmbhatt H.N. Wehland J. Rohde M. Timmis K.N. Infect. Immun. 1992; 60: 3345-3359Crossref PubMed Google Scholar). After preparation of periplasmic extracts, these were loaded on a QFF column (Pharmacia) and eluted by a linear NaCl gradient in 20 mmTris/HCl, pH 7.5. Depending on the construction, recombinant B-fragments eluted between 120 and 400 mm. B-fragment-containing fractions were dialyzed against 20 mmTris/HCl, pH 7.5, reloaded on a Mono Q column (Pharmacia), and eluted as before. The resulting proteins, estimated to be 95% pure by SDS-polyacrylamide gel electrophoresis, were stored at −80 °C until use. For iodination, 25 μg (for B-Glyc-KDEL and B-Glyc-KDELGL) or 100 μg (for B-fragment) of purified protein in elution buffer was treated with 200 μCi (B-fragment) or 500 μCi (B-Glyc-KDEL and B-Glyc-KDELGL) iodine (16.9 mCi/μg, Amersham Corp.) on a single IODO-BEAD (Pierce) according to the manufacturer's instructions. Proteins were labeled to specific activities of about 500 cpm/ng (B-fragment) or 5,000 cpm/ng (B-Glyc-KDEL and B-Glyc-KDELGL). Incorporated label was removed on PD10 gel filtration columns (Pharmacia). For metabolic sulfate labeling 4 × 105 HeLa cells were kept at 37 °C for 1 h in minimal essential medium without sulfate. After binding of B-Glyc-Sulf-KDEL to cells (see below), cells were incubated at 37 °C for 2 h in sulfate-free Hanks' balanced saline solution (Life Technologies, Inc.) containing 2 mCi/ml [35S]sulfate (1,000 Ci/mmol, Amersham). After labeling, cells were either placed on ice for direct immunoprecipitation or rinsed three times with chase medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum) before being incubated for 8 h in chase medium, followed by immunoprecipitation. For immunoprecipitation, cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.5% SDS, 1 mm phenylmethylsulfonyl fluoride, mixture of protease inhibitors containing leupeptine, chymostatin, pepstatin, antipain, and aprotinin in PBS). B-Glyc-Sulf-KDEL was immunoprecipitated using the monoclonal antibody 13C4 and protein A-Sepharose. Immunoprecipitates were washed three times in lysis buffer and once in 50 mm Tris/HCl, pH 7.5, boiled in sample buffer, and analyzed as described below. 1 × 105 cells were placed on ice and washed once with ice-cold PBS. The indicated concentrations of recombinant B-fragments (for Scatchard analysis, concentrations from 0.05 to 2 μm; for most other experiments, 50 nm) were added in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. After 90 min (for Scatchard analysis, incubation in presence of 10 mm HEPES, pH 7.2) or 45 min on ice, the cells were washed three times with ice-cold PBS. For Scatchard analysis, cells were lysed in 0.1 m KOH. Cell-associated radioactivity and radioactivity in the culture medium and wash solutions were counted, and binding data were obtained as published (25Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17806) Google Scholar). Iodinated B-Glyc-KDEL or B-Glyc-KDELGL was bound to 1 × 105 HeLa cells as described above. After incubation at 37 °C for the indicated periods of time cells were washed three times with PBS and lysed in SDS sample buffer. Samples were run on 10–20% polyacrylamide-SDS gradient gels, analyzed by autoradiography, and quantified with a PhosphorImager (Molecular Dynamics) using the ImageQuant software. In each experiment, the percentage of glycosylated protein was determined. The treatment with endoglycosidase H and peptideN-glycosidase F was done as described previously (26Martinez O. Schmidt A. Salaméro J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). In some experiments, internalization of modified B-fragments was performed in the presence of 1 μg/ml tunicamycin (added 1 h before the B-fragments) or 1 mm DMM (Boehringer Mannheim). 60 μg of recombinant B-fragments in 20 mm HEPES, pH 7.4, 150 mm NaCl, were added to 250 mmNaHCO3 and a 10-fold molar excess of DTAF (Sigma) and incubated by end-over-end rotation for 30 min at room temperature. 0.2 mm NH4Cl was added, and coupled protein was purified on PD10 columns. 0.7 × 105 HeLa cells, grown on 12-mm round glass coverslips, were incubated with 1 μg/ml DTAF-labeled recombinant B-fragments as described above. After the indicated internalization periods, cells were fixed with 3% paraformaldehyde for 10 min, permeabilized with saponin, stained with the indicated primary and secondary antibodies, and mounted, as described previously (26Martinez O. Schmidt A. Salaméro J. Hoflack B. Roa M. Goud B. J. Cell Biol. 1994; 127: 1575-1588Crossref PubMed Scopus (221) Google Scholar). Confocal laser scanning microscopy and immunofluorescence analysis were performed using a TCS4D confocal microscope based on a DM microscope interfaced with an argon/krypton laser. Simultaneous double fluorescence acquisitions were performed using the 488 nm and the 568 nm laser lines to excite fluorescein isothiocyanate and Texas Red dyes using a 63 × oil immersion Neofluar objective (NA = 1.4). The fluorescence was selected with appropriate double fluorescence dichroic mirror and band pass filters and measured with blue-green-sensitive and red side-sensitive one photomultipliers. B-Glyc-KDEL binding was performed on HeLa cells grown on 50-cm2 round tissue culture plates (1 × 107 cells), as described above. Cells were subsequently incubated at 37 °C as indicated, fixed in the culture dishes by adding to the medium an equivalent volume of 4% paraformaldehyde in 0.2 m sodium phosphate buffer, pH 7.4 for 1 h, and then further fixed with a fresh 2% paraformaldehyde solution in 0.1 sodium phosphate buffer for 1 more hour. Cells were collected by careful scraping and processed for cryosectioning according to Kleijmeer et al. (27Kleijmeer M.J. Raposo G. Geuze H.J. Methods: Companion to Methods Enzymol. 1996; 10: 191-207Crossref Scopus (48) Google Scholar). The cryosections were retrieved with a 1/1 solution of 2.3 m sucrose and 2% methyl cellulose according to Liou et al. (28Liou W. Geuze H.J. Slot J.W. Histochem. Cell Biol. 1996; 106: 41-58Crossref PubMed Scopus (437) Google Scholar). Immunogold labeling with the monoclonal antibody 13C4 (diluted 1/250) was performed with a rabbit anti-mouse linker antibody (Dako) and protein-A gold conjugate (purchased from Dr. J. W. Slot, Utrecht University). Double labeling experiments were performed as described previously (29Slot J.W. Geuze H.J. Gigengack S. Lienhard G.E. James D.E. J. Cell Biol. 1991; 113: 123-135Crossref PubMed Scopus (712) Google Scholar) using the rabbit polyclonal anti-galactosyltransferase antibody (O14 affinity-purified serum, diluted 1/100) as first primary antibody and 13C4 as the second one, diluted 1/250. Bacterial Shiga toxin is composed of two polypeptidic chains, termed A-fragment and B-fragment (14O'Brien A.D. Tesh V.L. Donohue-Rolfe A. Jackson M.P. Olsnes S. Sandvig K. Lindberg A.A. Keusch G.T. Curr. Top. Microbiol. Immunol. 1992; 180: 65-94Crossref PubMed Scopus (328) Google Scholar). Previous studies have shown that the whole toxin as well as isolated B-fragment are transported from the plasma membrane to the ER of toxin sensitive cells (15Garred O. Dubinina E. Holm P.K. Olsnes S. van Deurs B. Kozlov J.V. Sandvig K. Exp. Cell Res. 1995; 218: 39-49Crossref PubMed Scopus (60) Google Scholar, 16Khine A.A. Lingwood C.A. J. Cell. Physiol. 1994; 161: 319-332Crossref PubMed Scopus (69) Google Scholar, 17Sandvig K. Garred O. Prydz K. Kozlov J.V. Hansen S.H. van Deurs B. Nature. 1992; 358: 510-512Crossref PubMed Scopus (378) Google Scholar, 18Sandvig K. Ryd M. Garred O. Schweda E. Holm P.K. van Deurs B. J. Cell Biol. 1994; 126: 53-64Crossref PubMed Scopus (153) Google Scholar). To quantify this transport by monitoring the appearance of Shiga toxin B-fragment in the ER, anN-glycosylation site was added to the carboxyl terminus of the protein (Fig. 1 A). The carboxyl terminus of B-fragment was chosen since it sticks out of the globular conformation of the protein (30Fraser M.E. Chernaia M.M. Kozlov Y.V. James M.N. Nat. Struct. Biol. 1994; 1: 59-64Crossref PubMed Scopus (260) Google Scholar, 31Stein P.E. Boodhoo A. Tyrrell G.J. Brunton J.L. Read R.J. Nature. 1992; 355: 748-750Crossref PubMed Scopus (261) Google Scholar). N-Glycosylation starts in the ER by the addition of a core oligosaccharyl group from membrane-bound dolichol to an Asn-Xaa-Ser/Thr acceptor sequence on newly synthesized proteins (32Roth J. Biochim. Biophys. Acta. 1987; 906: 405-436Crossref PubMed Scopus (215) Google Scholar). Further modifications ofN-linked oligosaccharides occur only if this basic sugar backbone is present. B-fragment does not carry a KDEL motif (33Kozlov Yu V. Kabishev A.A. Lukyanov E.V. Bayev A.A. Gene ( Amst. ). 1988; 67: 213-221Crossref PubMed Scopus (59) Google Scholar, 34Seidah N.G. Donohue-Rolfe A. Lazure C. Auclair F. Keusch G.T. Chretien M. J. Biol. Chem. 1986; 261: 13928-13931Abstract Full Text PDF PubMed Google Scholar, 35Strockbine N.A. Jackson M.P. Sung L.M. Holmes R.K. O'Brien A.D. J. Bacteriol. 1988; 170: 1116-1122Crossref PubMed Google Scholar). Therefore, to favor its retention in the ER, the KDEL peptide was appended to the carboxyl terminus of the protein (Fig. 1 A). We also constructed a recombinant B-fragment carrying the KDELGL peptide (Fig. 1 A), which was previously found to be inactive with respect to KDEL-receptor interaction (6Miesenbock G. Rothman J.E. J. Cell Biol. 1995; 129: 309-319Crossref PubMed Scopus (112) Google Scholar). We will refer in this study to the recombinant B-fragments as B-Glyc-KDEL and B-Glyc-KDELGL ("B" for B-fragment, "Glyc" for N-glycosylation site, and "KDEL" or "KDELGL" for KDEL or KDELGL peptides). It has been shown previously that Shiga toxin binds to the glycolipid Gb3, which is expressed by toxin-sensitive cells (36Lingwood C.A. Trends Microbiol. 1996; 4: 147-153Abstract Full Text PDF PubMed Scopus (235) Google Scholar) such as HeLa cells. To test whether the carboxyl-terminal modifications made on the B-fragment influenced its interaction with cells, we first compared binding to and internalization into HeLa cells of B-Glyc-KDEL and B-Glyc-KDELGL with that of the wild type B-fragment. Recombinant B-fragments were purified from E. coli and iodinatedin vitro (see "Materials and Methods"). Unlabeled wild type B-fragment and B-Glyc-KDEL competed equally well with radiolabeled B-Glyc-KDEL for binding to HeLa cells (Fig. 2). Half-maximal competition was observed at 300 nm cold competitor protein. Scatchard analysis showed that the binding constants of wild type B-fragment, of B-Gly-KDEL, and of B-Gly-KDELGL were comparable (Table I). We calculated that 1–2 × 106 binding sites were present per HeLa cell (Table I), in good agreement with the literature (37Eiklid K. Olsnes S. J. Recept. Res. 1980; 1: 199-213Crossref PubMed Scopus (39) Google Scholar). All three proteins were also found to be equally well internalized into HeLa cells (not shown). Altogether, the results indicated that the modifications of the carboxyl terminus of B-fragment did not significantly change its binding and uptake properties.Table IScatchard analysis of binding of iodinated wild type B-fragment and B-Glyc-KDEL to HeLa cellsConstructionK DBinding sites (×106/cell)nmB-fragment250 (±17; n= 2)1.5 (±0.1; n = 2)B-Glyc-KDEL100 (±5;n = 3)1.2 (±0.3; n = 3)B-Glyc-KDELGL238 (±12; n = 3)1.9 (±0.1;n = 3) Open table in a new tab Iodinated B-Glyc-KDEL or B-Glyc-KDELGL were first bound for 45 min to HeLa cells on ice. After binding, the cells were incubated for various lengths of time at 37 °C (Fig. 3 A). At the end of each incubation period, cells were lysed, and proteins were separated by SDS-polyacrylamide gel electrophoresis. At time 0 (45 min of binding on ice), single fragments migrating with an apparent molecular mass of 9.5 kDa and corresponding to the expected size of B-Glyc-KDEL and B-Glyc-KDELGL were observed (Fig. 3 A). B-Glyc-KDEL migrated slightly faster than B-Glyc-KDELGL (Fig.3 A), consistent with the fact that B-Glyc-KDELGL is two amino acids longer than B-Glyc-KDEL. Internalization of B-Glyc-KDEL led to the appearance of further bands (Fig. 3 A,left). After 1, 2, or 4 h, one species (Fig.3 A, triangle) and after 15 h, two species (Fig. 3 A, triangle and asterisk) with a lower electrophoretic mobility than the original B-Glyc-KDEL were observed. As shown in Fig. 3 B, their appearance was strongly inhibited by treatment of cells with tunicamycin, an inhibitor ofN-glycosylation. In addition, these bands were lost after treatment of samples with endoglycosidase H or peptideN-glycosidase F, two N-glycosylation-specific glycosidases (Fig. 3 B). These observations suggest that the upper bands represent N-glycosylated B-Glyc-KDEL. The fact that they remained endoglycosidase H-sensitive indicated that B-Glyc-KDEL reached the ER and then stayed associated with it. The appearance of the second species of N-glycosylated B-Glyc-KDEL (Fig. 3 A, asterisk) was inhibited by DMM (Fig. 3 C), an inhibitor of mannosidase I, indicating that this species resulted from the trimming of the primary glycosylation product of B-Glyc-KDEL (Fig. 3 A,triangle) by cis-Golgi-specific mannosidase activity. B-Glyc-KDEL thus cycles between ER and cis-Golgi compartments, as described for other resident ER proteins (1Dean N. Pelham H.R. J. Cell Biol. 1990; 111: 369-377Crossref PubMed Scopus (126) Google Scholar, 5Pelham H.R. Hardwick K.G. Lewis M.J. EMBO J. 1988; 7: 1757-1762Crossref PubMed Scopus (189) Google Scholar). The same results on glycosylation of B-Glyc-KDEL were obtained in A431 cells and Vero cells (not shown). The incubation of HeLa cells with B-Glyc-KDELGL gave a different pattern. Multiple bands with a lower electrophoretic mobility than unmodified B-Glyc-KDELGL were observed, giving the appearance of a smear (Fig. 3 A, open bracket). Some of them were found to be endoglycosidase H-resistant (not shown). In addition, the appearance of the upper bands was inhibited by DMM (Fig.3 C). These observations suggest that B-Glyc-KDELGL was also transported to the ER in HeLa cells where it was glycosylated. In contrast to B-Glyc-KDEL, B-Glyc-KDELGL seemed to be able to move in the anterograde direction up to cis/medial Golgi and acquire complex carbohydrates (endoglycosidase H resistance). The unequal behavior of B-Glyc-KDEL and B-Glyc-KDELGL was also observed in morphological studies (see below). In addition to slower migrating glycosylation products, faster migrating bands were observed after incubation of B-Glyc-KDEL or B-Glyc-KDELGL with cells (Fig. 3 A, circles). These proteins were insensitive to tunicamycin, endoglycosidase H, and peptide N-glycosidase F treatment (Fig. 3 B) and could represent degradation products. In this respect, it is interesting to note that even after 15 h of incubation in cells, less than 3% of internalized B-Glyc-KDEL and B-Glyc-KDELGL became trichloroacetic acid-soluble (not shown). This indicates that if the lower bands are degradation products, these remain stably associated with cells. To quantify the arrival of modified B-fragments in the ER, autoradiographs were scanned using a PhosphorImager (Fig.3 D). To allow an accurate quantification of the glycosylated bands, especially for B-Glyc-KDELGL, internalization of modified B-fragments was performed in the presence of DMM. Glycosylation of B-Glyc-KDEL increased linearly with incubation time and reached 22% of the total cell-associated protein after a 15-h incubation (Fig.3 D). Similar results were obtained for cells incubated without DMM (not shown). Interestingly, glycosylation of B-Glyc-KDELGL was only slightly less efficient than that of B-Glyc-KDEL (Fig.3 D). In addition, the kinetics of glycosylation was not markedly modified. This indicates that the functional KDEL motif is important for the retention of the protein within the ER but not for its retrograde transport. To characterize the intracellular pathway followed by B-Glyc-KDEL or B-Glyc-KDELGL from the plasma membrane to the ER, recombinant proteins were covalently linked to the fluorophore DTAF. After 15 min at 37 °C, the bulk of B-Glyc-KDEL was found associated with the plasma membrane and endosomes, as shown by its colocalization with the transferrin receptor (Fig. 4,A and B). After 45 min, the protein became detectable in the Golgi apparatus where it colocalized with Rab6, a GTPase associated with Golgi and TGN membranes (38Goud B. Zahraoui A. Tavitian A. Saraste J. Nature. 1990; 345: 553-556Crossref PubMed Scopus (252) Google Scholar, 39Antony C. Cibert C. Geraud G. Santa Maria A. Maro B. Mayau V. Goud B. J. Cell Sci. 1992; 103: 785-796Crossref PubMed Google Scholar) (Fig. 4,C and D). After 4 h at 37 °C, B-Glyc-KDEL displayed a typical ER staining and colocalized with the signal sequence receptor protein (Fig. 4, E andF). In contrast to B-Glyc-KDEL, B-Glyc-KDELGL was mainly localized in the Golgi region after incubation of cells for 1 h, 4 h (not shown), or 15 h at 37 °C (Fig. 4, G andH). The same result was obtained for DTAF-labeled wild type B-fragment (not shown), which is in good agreement with previously published experiments on B-fragment transport to the Golgi apparatus (40Kim J.H. Lingwood C.A. Williams D.B. Furuya W. Manolson M.F. Grinstein S. J. Cell Biol. 1996; 134: 1387-1399Crossref PubMed Scopus (121) Google Scholar). However, a fraction of B-Glyc-KDELGL was present in the ER, as documented by colocalization with signal sequence receptor protein (Fig. 4, G and H). These data are consistent with the hypothesis that B-Glyc-KDELGL is transported to the ER and that the protein can then move in the anterograde direction up to the Golgi apparatus (see above; Fig. 3 A). In agreement with the literature, we noticed that the intensity of B-Glyc-KDEL labeling showed some heterogeneity from cell to cell in the same population (17Sandvig K. Garred O. Prydz K. Kozlov J.V. Hansen S.H. van Deurs B. Nature. 1992; 358: 510-512Crossref PubMed Scopus (378) Google Scholar, 18Sandvig K. Ryd M. Garred O. Schweda E. Holm P.K. van Deu
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