The Protein-tyrosine Phosphatase CD45 Reaches the Cell Surface via Golgi-dependent and -independent Pathways
2002; Elsevier BV; Volume: 277; Issue: 52 Linguagem: Inglês
10.1074/jbc.m209075200
ISSN1083-351X
AutoresTroy A. Baldwin, Hanne L. Ostergaard,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoCD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at ∼15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitiveN-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface. CD45 is a receptor protein-tyrosine phosphatase essential for T cell development and lymphocyte activation. It is highly glycosylated, with multiple isoforms and glycoforms expressed on the cell surface depending on the cell type and stage of differentiation. Interestingly, we found two pools of newly synthesized CD45 expressed on plasma membrane, one of which arrived by 5 min after synthesis. The remaining pool of CD45 was fully glycosylated and began to arrive at the cell surface at ∼15 min. The rapidly expressed population of CD45 possessed exclusively endoglycosidase H-sensitiveN-linked carbohydrate. Additionally, this rapidly expressed pool of CD45 appeared on the cell surface in a brefeldin A (BFA)-insensitive manner, suggesting that it reached the cell surface independent of the Golgi complex. The remaining CD45 trafficked through the Golgi complex, and transport proceeded via a BFA-sensitive mechanism. These data suggest that CD45 is able to reach the cell surface via two distinct routes. The first is a conventional Golgi-dependent pathway that allows fully processed CD45 to be expressed. The second utilizes an ill defined mechanism that is independent of the Golgi, is BFA-resistant, and allows for the expression of CD45 with immature carbohydrate on the cell surface. endoplasmic reticulum glucosidase II trans-Golgi network major histocompatibility complex endoglycosidase H phosphate-buffered saline brefeldin A fluorescence-activated cell sorter endoplasmic reticulum-Golgi intermediate compartment coat protein complex cystic fibrosis transmembrane conductance regulator The transmembrane protein-tyrosine phosphatase CD45 is required for both thymocyte development and T cell activation (1Byth K.F. Conroy L.A. Howlett S. Smith A.J. May J. Alexander D.R. Holmes N. J. Exp. Med. 1996; 183: 1707-1718Crossref PubMed Scopus (356) Google Scholar, 2Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (659) Google Scholar). CD45 exerts its effects, at least in part, by regulating the phosphorylation state of Src family kinases through the dephosphorylation of a negative regulatory carboxyl-terminal tyrosine residue (3Thomas M.L. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 4Ashwell J.D. D'Oro U. Immunol. Today. 1999; 20: 412-416Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition to the cytoplasmic phosphatase domains, CD45 contains a large extracellular region. Three alternatively spliced exons reside within the external domain of CD45 and contain numerous sites for potentialO-linked carbohydrate additions. These exons are developmentally regulated with respect to usage in T cells; and therefore, cells at different developmental stages have the potential to express vastly different forms of CD45. In addition to theO-linked carbohydrate found in the alternatively spliced sequences, there are numerous potential N-linked carbohydrate attachment sites (5Thomas M.L. Annu. Rev. Immunol. 1989; 7: 339-369Crossref PubMed Google Scholar). These N-linked carbohydrate additions have been demonstrated to be important for both cell-surface expression and stability of CD45 (6Pulido R. Sanchez-Madrid F. Eur. J. Immunol. 1992; 22: 463-468Crossref PubMed Scopus (25) Google Scholar). In addition, we have recently shown that the composition of the CD45 N-linked carbohydrate is developmentally regulated, possibly through the action of the endoplasmic reticulum (ER)1 enzyme glucosidase II (GII) (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar). Intriguingly, no typical cell-surface ligand for the extracellular domain of CD45 has been identified; however, several lectins have been demonstrated to bind CD45 carbohydrate (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar, 8Stamenkovic I. Sgroi D. Aruffo A., Sy, M.S. Anderson T. Cell. 1991; 66: 1133-1144Abstract Full Text PDF PubMed Scopus (314) Google Scholar, 9Sgroi D. Koretzky G.A. Stamenkovic I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4026-4030Crossref PubMed Scopus (81) Google Scholar, 10Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811Crossref PubMed Google Scholar, 11Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (942) Google Scholar, 12Uemura K. Yokota Y. Kozutsumi Y. Kawasaki T. J. Biol. Chem. 1996; 271: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The intracellular transport of proteins from the ER to the cell surface is a tightly controlled process involving the coordinated action of many enzymes and proteins (13Lippincott-Schwartz J. Roberts T.H. Hirschberg K. Annu. Rev. Cell Dev. Biol. 2000; 16: 557-589Crossref PubMed Scopus (384) Google Scholar). For the most part, as a protein moves through the secretory pathway, a level of control is exerted at each stage of the transport process, from protein folding and vesicle budding at the ER to movement through the Golgi stacks and finally sorting at the trans-Golgi network (TGN) en route to its final destination (14Roche P.A. Immunity. 1999; 11: 391-398Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In many cases, this regulation is necessary for proper function of the protein. For example, transport of Class I and II major histocompatibility complex (MHC) antigen-presenting proteins as well as CD1 follows different routes to the cell surface, and those specific routes are necessary to ensure that the appropriate antigens are loaded into their peptide- or glycolipid-binding grooves (15Briken V. Jackman R.M. Dasgupta S. Hoening S. Porcelli S.A. EMBO J. 2002; 21: 825-834Crossref PubMed Scopus (79) Google Scholar, 16Pieters J. Biol. Chem. Hoppe-Seyler. 1997; 378: 751-758PubMed Google Scholar, 17Pamer E. Cresswell P. Annu. Rev. Immunol. 1998; 16: 323-358Crossref PubMed Scopus (873) Google Scholar). For CD45, it is clear that cell-surface expression is required for proper function and that the N-linked carbohydrate on CD45 plays a role in this process (6Pulido R. Sanchez-Madrid F. Eur. J. Immunol. 1992; 22: 463-468Crossref PubMed Scopus (25) Google Scholar). Recently, mannose-binding lectin has been shown to bind cell-surface CD45, which indicates that CD45 is able to escape complete carbohydrate processing, leaving immature, high mannose carbohydrate (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar, 12Uemura K. Yokota Y. Kozutsumi Y. Kawasaki T. J. Biol. Chem. 1996; 271: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Alternative trafficking routes are one possible mechanism CD45 could employ to reach the plasma membrane without complete carbohydrate processing. With the recent examples of carbohydrate influencing biological function such as dendritic cell-specific ICAM-grabbing non-integrin interactions (18Geijtenbeek T.B. Torensma R. van Vliet S.J. van Duijnhoven G.C. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Abstract Full Text Full Text PDF PubMed Scopus (1450) Google Scholar, 19Geijtenbeek T.B.H. Krooshoop J.E.B. Bleijs D.A. van Vliet S.J. van Duijnhoven G.C.F. Brabovsky V. Alon R. Figdor C.G. van Kooyk Y. Nat. Immunol. 2000; 1: 353-357Crossref PubMed Scopus (432) Google Scholar), CD8-MHC interactions (20Daniels M.A. Devine L. Miller J.D. Moser J.M. Lukacher A.E. Altman J.D. Kavathas P. Hogquist K.A. Jameson S.C. Immunity. 2001; 15: 1051-1061Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 21Moody A.M. Chui D. Reche P.A. Priatel J.J. Marth J.D. Reinherz E.L. Cell. 2001; 107: 501-512Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), and T cell receptor clustering (22Demetriou M. Granovsky M. Quaggin S. Dennis J.W. Nature. 2001; 409: 733-739Crossref PubMed Scopus (739) Google Scholar), as well as the limited information on CD45 trafficking in general, we set out to determine the overall trafficking patterns of CD45. By examining the transport of CD45 to the plasma membrane, we hoped to gain insight into the mechanism of expression of CD45 bearing mannose-binding lectin ligands. Our results indicate that there is a pool of CD45 that is very rapidly expressed on the cell surface after synthesis. It appears that two different mechanisms exist that allow CD45 to reach the cell surface: one involving the Golgi complex, resulting in endoglycosidase H (Endo H)-resistant carbohydrate modification; and the other independent of the Golgi, resulting in the maintenance of exclusively Endo H-sensitive carbohydrate on the cell surface. These data support the existence of a transport pathway where cargo can reach the cell surface extremely rapidly, without the requirement of the Golgi complex. The T lymphoma cell line BW5147 (referred to below as BW) was maintained as previously described (23Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1995; 270: 2313-2319Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The CD45RO-expressing fibroblast cell line ψ2 50.5 was provided by Dr. Pauline Johnson (University of British Columbia, Vancouver, British Columbia, Canada) (24Johnson P. Greenbaum L. Bottomly K. Trowbridge I.S. J. Exp. Med. 1989; 169: 1179-1184Crossref PubMed Scopus (60) Google Scholar). The anti-pan CD45 extracellular domain monoclonal antibody I3/2 was described previously (23Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1995; 270: 2313-2319Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The anti-Class I MHC Db antiserum H137 (25Maksymowych W.P. Ikawa T. Yamaguchi A. Ikeda M. McDonald D. Laouar L. Lahesmaa R. Tamura N. Khuong A., Yu, D.T. Kane K.P. Infect. Immun. 1998; 66: 4624-4632Crossref PubMed Google Scholar) was kindly provided by Dr. Kevin Kane (University of Alberta). Rabbit antisera H2 and J37, specific for GIIβ and the intracellular region of CD45, respectively, were previously described (26Baldwin T.A. Gogela-Spehar M. Ostergaard H.L. J. Biol. Chem. 2000; 275: 32071-32076Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Rabbit anti-Pyk-2 antiserum was previously described (27Berg N.N. Ostergaard H.L. J. Immunol. 1997; 159: 1753-1757PubMed Google Scholar). Anti-GIIα antiserum was purchased from Stressgen Biotechnologies Corp. (Vancouver). Cell-surface biotinylation was performed as previously described (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar). Briefly, cells were biotinylated with 50 μl of 10 mm sulfosuccinimidobiotin (Pierce)/5 × 107 cells/ml in phosphate-buffered saline (PBS) for 20 min on ice. Reactions were quenched by washing cells twice with PBS containing 5 mm glycine. All cells were lysed at a density of 5 × 107/ml in 0.5% Nonidet P-40 (Pierce)/Tris-buffered saline buffer (lysis buffer) and incubated for 20 min on ice. Post-nuclear supernatants were incubated with I3/2-coupled Sepharose 4B for 1–2 h or with 10 μl/ml rabbit antiserum, followed by capture of the immune complexes with protein A-Sepharose 4B. Biotinylated proteins were isolated by incubation with streptavidin-agarose for 1–2 h at 4 °C. Immunoprecipitates and streptavidin pull-downs were washed three times with radioimmune precipitation assay buffer. After washing, the immunoprecipitates were boiled in reducing sample buffer. Immunoprecipitates were treated with Endo H (Calbiochem) in PBS containing 1% Nonidet P-40, 0.1% SDS, and 1% 2-mercaptoethanol for 16 h at 33 °C. Cells were washed twice with PBS prior to depletion of intracellular methionine by incubation for 30 min at 37 °C with methionine-free RPMI 1640 medium (Invitrogen). Cells were then pulsed for 10 min at 15 °C or for 5 min at 37 °C (pulse conditions are indicated for each figure) with 0.5 mCi/ml Tran35S-label (ICN Biomedicals) at 5 × 107/ml. Cells were washed twice with ice-cold unlabeled complete medium (containing methionine) prior to initiation of the chase. Cells were chased at 37 °C (or as indicated) in complete medium, followed by washing with PBS prior to lysis. Where indicated, the chase medium was supplemented with 10 mm methionine. All cells were kept on ice after the chase prior to isolation of the cell-surface protein as described below to prevent antibody or biotin internalization. For specific isolation of cell-surface proteins, either an antibody or biotinylation method was employed. For isolation of cell-surface protein by the antibody method, cells were incubated for 20 min with 20 μg/ml I3/2 or 10 μl/ml rabbit antiserum at 4 °C, followed by washing three times with PBS. Cells (5 × 106) were then lysed with 500 μl of 2.5 × 107/ml unlabeled lysate. Immune complexes were recovered with secondary antibody-coated protein A-Sepharose 4B. For isolation of cell-surface CD45 by biotinylation, cells were surface-biotinylated as described above and lysed at 2.5 × 107/ml in lysis buffer, followed by total CD45 immunoprecipitation. Captured CD45 was released by boiling for 2 × 5 min in 50 μl of 2% SDS-containing Tris-buffered saline. Eluent was diluted to 1 ml with lysis buffer. Biotinylated CD45 was isolated with immobilized streptavidin as described above. Cell-surface proteins were selectively isolated by the antibody method unless otherwise stated. All immunoprecipitates and pull-downs were washed three times with radioimmune precipitation assay buffer prior to boiling with reducing sample buffer. In the cases where brefeldin A (BFA) was used to block protein trafficking, cells were preincubated with 2 μg/ml BFA for 30 min at 37 °C. BFA was also present during the pulse-chase at 2 μg/ml. Proteins were resolved on polyacrylamide gels and transferred to Immobilon (polyvinylidene difluoride; Millipore Corp., Bedford, MA) as described previously (23Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1995; 270: 2313-2319Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). For separation under nonreducing conditions, 2-mercaptoethanol was omitted from the sample buffer. Autoradiography was performed with the BioMax TranScreen intensifying system (Eastman Kodak Co.). Western blot analysis was conducted with the indicated antiserum followed by horseradish peroxidase-conjugated protein A (Pierce) and visualized by ECL (PerkinElmer Life Sciences). Densitometry was performed using NIH Image Version 1.62 software. Cells (1 × 106) were incubated with 10 μl/ml antiserum H2 for 20 min on ice, followed by two washes with PBS containing 0.1% serum. For detection of bound antibody, cells were further incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody for an additional 20 min on ice. Cells were then fixed in 1% paraformaldehyde before analysis. Because CD45 is capable of reaching the cell surface with incompletely processed carbohydrate (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar, 12Uemura K. Yokota Y. Kozutsumi Y. Kawasaki T. J. Biol. Chem. 1996; 271: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), we wished to follow the processing of CD45 carbohydrate through the secretory pathway using the acquisition of Endo H resistance as a marker of protein location. Treatment of newly synthesized CD45 with Endo H after various chase times indicated that CD45 began to acquire Endo H resistance ∼15 min after synthesis and that 80% of newly synthesized CD45 was Endo H-resistant after 60 min (Fig. 1). Class I MHC showed a lag period before acquiring Endo H resistance (Fig. 1). This lag phase in glycoprotein trafficking seen with Class I MHC may reflect the requirement for peptide loading, β2-microglobulin association, and transport to the Golgi before Class I begins to achieve Endo H resistance. It should be noted at this point that CD45 never achieved full Endo H resistance, with ∼50% of its carbohydrate remaining Endo H-sensitive (Fig. 1). In addition to determining the rate of acquisition of Endo H resistance, another key parameter in the trafficking of CD45 is the time required to reach the cell surface. By ascertaining the time required to reach the plasma membrane, we can observe whether or not CD45 traffics directly from the Golgi to the cell surface, or if a route through other compartments occurs. Initial experiments performed to address the time required for newly synthesized CD45 to reach the plasma membrane indicated that a population of newly synthesized CD45 was able to traffic to the surface during a 5-min pulse at 37 °C (data not shown). This time to cell-surface expression seems to be extremely rapid compared with the kinetics reported for the majority of other cell-surface proteins. However, Nori and Stallcup (28Nori M. Stallcup M.R. Mol. Cell. Biol. 1988; 8: 833-842Crossref PubMed Scopus (2) Google Scholar) used CD45 as a control in a pulse-chase experiment and reported similar results with respect to rapid CD45 cell-surface expression. To more accurately determine the time required for newly synthesized CD45 to reach the cell surface, a modification to the previous pulse-chase protocol was necessary. A pulse condition was needed that prevented or substantially slowed bulk protein transport, but still allowed for sufficient incorporation of metabolic label. Incubation of cells at 15 °C has previously been demonstrated to block protein transport at the ER-Golgi intermediate compartment (ERGIC) (29Saraste J. Kuismanen E. Cell. 1984; 38: 535-549Abstract Full Text PDF PubMed Scopus (350) Google Scholar); therefore, pulsing cells with [35S]methionine at 15 °C instead of 37 °C should slow protein transport enough to reveal a true zero chase time for cell-surface expression. Using this new protocol, no radiolabeled CD45 was detected on the cell surface at time 0 (Fig. 2 A). By 5 min, a lower molecular weight population of newly synthesized CD45 appeared at the plasma membrane, whereas a higher molecular weight population reached the cell surface at ∼15 min. The amount of newly synthesized, higher molecular weight CD45 isolated from the cell surface increased through 60 min, whereas the amount of the lower molecular weight population remained fairly constant after 15 min. As determined by densitometric analysis, at 60 min, the lower molecular weight population of newly synthesized cell-surface CD45 composed ∼20% of the total newly synthesized cell-surface material; and in examining the steady-state levels of surface CD45, the lower form was ∼20% of the total (Fig. 2 A). We also observed that the total amount of newly synthesized CD45 increased over the chase time, which raises the possibility that labeled CD45 continues to be synthesized over the chase or that, because the antibody used to isolate CD45 is conformation-dependent, this increase may reflect an increase in the amount of folded CD45. Because the inclusion of 10 mm methionine during the chase did not prevent the increase in labeled CD45 isolated over the chase (Fig. 2 A,expt. 2), we suspect that the increase in labeled CD45 recovered is due to an increased ability of the antibody to recognize CD45 over the chase time. It still remains possible that, during the cell-surface isolation of newly synthesized CD45, we were recovering internal pools of labeled CD45 nonspecifically. This may be particularly problematic at the early time points. For a number of reasons, we feel that this possibility is unlikely. Perhaps the most compelling argument against this possibility is that, using the same isolation protocol with a fibroblast cell line transfected with CD45, we were unable to recover newly synthesized cell-surface CD45 until 30 min, even though a significant amount of newly synthesized CD45 could be recovered over the chase time (Fig. 2 B). In fact, with this cell line, cells were pulsed for 5 min at 37 °C before initiating the chase; and even with this pulse procedure, a rapidly expressed population of CD45 was not detected (Fig. 2 B). Additionally, there appears to be only one glycoform of CD45 isolated from these cells, in contrast to the two forms isolated from the BW cell line. Furthermore, using Class I MHC as a control for the protocol, the surface expression of newly synthesized Class I MHC followed trafficking kinetics similar to those previously reported (30Williams D.B. Swiedler S.J. Hart G.W. J. Cell Biol. 1985; 101: 725-734Crossref PubMed Scopus (83) Google Scholar, 31Williams D.B. Borriello F. Zeff R.A. Nathenson S.G. J. Biol. Chem. 1988; 263: 4549-4560Abstract Full Text PDF PubMed Google Scholar), with no newly synthesized Class I MHC being isolated from the cell surface until 30 min of chase time (Fig. 2 C). Finally, control mixing experiments in which unlabeled surface protein was isolated in the presence of labeled lysates showed that barely detectable levels of labeled material (significantly lower than the amount we detected at the early time points in Fig. 2 A) were isolated with the unlabeled cell-surface complexes (data not shown). Another published method to isolate cell-surface proteins involves surface biotinylation and isolation of the protein of interest, followed by streptavidin affinity enrichment (15Briken V. Jackman R.M. Dasgupta S. Hoening S. Porcelli S.A. EMBO J. 2002; 21: 825-834Crossref PubMed Scopus (79) Google Scholar, 32Hobman T.C. Woodward L. Farquhar M.G. J. Cell Biol. 1993; 121: 269-281Crossref PubMed Scopus (49) Google Scholar). Using the surface biotinylation approach to isolate cell-surface CD45, we obtained results similar to those attained with the surface antibody method described above (Fig. 3 A). Newly synthesized surface-biotinylated CD45 began to appear on the cell surface at 5 min and increased through to 60 min of chase time, with the existence of two different forms (Fig. 3 A). Between experiments and surface CD45 isolation protocols, the only variability in results occurred in the amount of the higher molecular weight form of CD45 isolated at 15 min. To control for possible nonspecific isolation of cell-surface CD45 by the biotinylation method, two controls were performed. First, a cytosolic protein-tyrosine kinase (Pyk-2) was immunoprecipitated from a surface-biotinylated lysate or a lysate biotinylated after detergent solubilization. Pyk-2 was found to be biotinylated only in the case where biotin was added post-lysis, not during the surface biotinylation procedure (Fig. 3 B). Second, non-biotinylated CD45 eluted from a CD45 immunoprecipitate was not captured by streptavidin-coated beads (Fig. 3 C). Therefore, it appears that there is a rapidly expressed population of CD45 in BW cells and that the mechanism used to express this form of CD45 does not exist in all cell types. Because bulk CD45 acquired Endo H resistance rapidly after synthesis and there were two different forms of CD45 expressed on the cell surface with different kinetics, we wished to determine whether both forms were in fact Endo H-resistant or if one was possibly Endo H-sensitive. To determine the carbohydrate status of the two different forms of newly synthesized cell-surface CD45, surface CD45 was isolated after pulse-chase and subjected to Endo H treatment. The rapidly expressed population of CD45 was entirely Endo H-sensitive, whereas the higher molecular weight form achieved its full Endo H resistance (Fig. 4). These data suggest that the higher molecular weight form is a mature glycoform of CD45 with fully processed carbohydrate, whereas the lower molecular weight form is an immature glycoform with unprocessed carbohydrate. Note again that the fully processed glycoform of CD45 still contains a significant fraction of Endo H-sensitive carbohydrate, but mature cell-surface CD45 always appears to contain this level of Endo H-sensitive carbohydrate in these cells (7Baldwin T.A. Ostergaard H.L. J. Immunol. 2001; 167: 3829-3835Crossref PubMed Scopus (22) Google Scholar). The finding that CD45 was capable of reaching the cell surface without complete processing of its carbohydrate raises the question of how this material traffics from the ER to the cell surface. The prevailing model of glycoprotein transport states that once a glycoprotein leaves the ER, it is transported through the ERGIC to the Golgi, where the carbohydrate is processed to a complex form, and finally to the cell surface. Our finding that the rapidly expressed pool of CD45 reached the cell surface with exclusively unprocessed N-linked carbohydrate suggests that this pool may by-pass the Golgi entirely en route to the cell surface. One of the most well characterized and commonly used methods to inhibit protein transport through the Golgi is treatment of cells with the fungal metabolite BFA. BFA interferes with the recruitment of the ADP-ribosylation factor-1 GTPase to COPI-coated membranes, effectively blocking protein transport through the prevention of ER export and subsequent Golgi redistribution (33Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (594) Google Scholar,34Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (578) Google Scholar). Treatment of cells with BFA effectively inhibited Class I MHC cell-surface expression and the trafficking of the higher molecular weight mature glycoform of CD45, indicating that the BFA-induced blockade was successful; however, it did not inhibit the transport of the rapidly expressed, lower molecular weight glycoform of CD45 to the cell surface (Fig. 5). Examination of the carbohydrate on newly synthesized CD45 after BFA treatment revealed that it contained entirely Endo H-sensitive carbohydrate, as expected (Fig. 5). These data suggest that the rapidly expressed population of CD45 reaches the cell surface independent of the Golgi complex and does not rely on a BFA-sensitive transport mechanism. As we did not observe increasing amounts of CD45 reaching the cell surface after BFA treatment, and the amount of cell-surface CD45 appeared fixed at ∼20% in the presence of BFA, the data suggest that there is an early commitment of a significant portion of CD45 to the conventional transport pathway. Apparently, once it enters the conventional pathway, CD45 cannot be expressed on the cell surface in the presence of BFA by the rapid alternative pathway. To further dissect the pathway used by the rapidly expressed pool of CD45, we took advantage of blocks at the ERGIC and TGN imposed by chasing proteins at 15 and 20 °C, respectively (29Saraste J. Kuismanen E. Cell. 1984; 38: 535-549Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 35Griffiths G. Pfeiffer S. Simons K. Matlin K. J. Cell Biol. 1985; 101: 949-964Crossref PubMed Scopus (282) Google Scholar, 36Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (761) Google Scholar). Performing the chase at 15 °C resulted in delayed and reduced expression of the lower molecular weight form of CD45, whereas this treatment completely prevented the surface expression of the higher molecular weight form of CD45 and Class I MHC, indicating that the blockade was successful (Fig. 6 A). Chasing cells at 20 °C after pulsing at 15 °C and isolation of cell-surface CD45 resulted in the appearance of the lower molecular weight form of CD45 beginning at 15 min and increasing through the entire chase time (Fig. 6 B). By chasing at 20 °C, both the higher molecular weight form of CD45 and Class I MHC were prevented from reaching the plasma membrane, indicating that the kinetic block was functional (Fig. 6 B). Examination of the remaining CD45 after the chase at 15 or 20 °C revealed that it still contained predominantly Endo H-sensitive carbohydrate (Fig. 6, A and B). The observation of reduced kinetics of acquisition of Endo H resistance is in accordance with previously published data (35Griffiths G. Pfeiffer S. Simons K. Matlin K. J. Cell Biol. 1985; 101: 949-964Crossref PubMed Scopus (282) Google Scholar). Therefore, it appears that a block of glycoprotein traffic through thecis-Golgi or at the TGN does not prevent the expression of the lower molecular weight form of CD45, but it does appear to delay its trafficking kinetics. Because CD45 and GII associate stably in the BW cells used for this study and a number of other putative resident ER proteins have been found on the cell surface of numerous cell types (37Arosa F.A. de Jesus O. Porto G. Carmo A.M. de Sousa M. J. Biol. Chem. 1999; 274: 16917-16922Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 38Mezghrani A. Courageot J. Mani
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