Role of COPI in Phagosome Maturation*

Received for publication, December 20, 1999, and in revised form, March 13, 2000Published, JBC Papers in Press, March 15, 2000, DOI 10.1074/jbc.M910068199

Roberto J. Botelho‡§, David J. Hackam‡, Alan D. Schreiber¶, and Sergio Grinstein‡i

From the ‡Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada and Departmentof Biochemistry of the University of Toronto, Toronto, Ontario M5G 1X8, Canada and the ¶Department of Medicine,University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4283

Phagosomes mature by sequentially fusing with endo-somes and lysosomes. Vesicle budding is presumed tooccur concomitantly, mediating the retrieval of plas-malemmal components and the regulation of phagoso-mal size. We analyzed whether fission of vesicles fromphagosomes requires COPI, a multimeric complexknown to be involved in budding from the Golgi andendosomes. The role of COPI was studied using ldlFcells, that harbor a temperature-sensitive mutation ine-COP, a subunit of the coatomer complex. These cellswere made phagocytic toward IgG-opsonized particlesby heterologous expression of human FcgRIIA recep-tors. Following incubation at the restrictive tempera-ture, e-COP was degraded in these cells and their Golgicomplex dispersed. Nevertheless, phagocytosis per-sisted for hours in cells devoid of e-COP. Retrieval oftransferrin receptors from phagosomes became ineffi-cient in the absence of e-COP, while clearance of theFcgRIIA receptors was unaffected. This indicates thatfission of vesicles from the phagosomal membrane in-volves at least two mechanisms, one of which requiresintact COPI. Traffic of fluid-phase markers and aggre-gated IgG-receptor complexes along the endocytic path-way was abnormal in e-COP-deficient cells. In contrast,phagosome fusion with endosomes and lysosomes wasunimpaired. Moreover, the resulting phagolysosomeswere highly acidic. Similar results were obtained inRAW264.7 macrophages treated with brefeldin A, whichprecludes COPI assembly by interfering with the acti-vation of adenosine ribosylation factor. These data indi-cate that neither phagosome formation nor maturationare absolutely dependent on COPI. Our findings implythat phagosomal maturation differs from endosomalprogression, which appears to be more dependent onCOPI-mediated formation of carrier vesicles.

Phagocytosis plays a key role in the host immune defense bysequestering invading microorganisms within vacuoles formedby invagination of the plasma membrane of neutrophils andmacrophages (1–3). Such vacuoles, known as phagosomes, un-dergo sequential fusion with early and late endosomes and

ultimately with lysosomes (4–8). Budding of vesicles from thephagosome is thought to occur in parallel with fusion (9–12),thereby maintaining the surface area of the phagosome approx-imately constant. Jointly, vesicular fusion and fission lead toremodeling of the phagosomal membrane and contents, a proc-ess known as phagosomal maturation (6). The final stage ofthis sequence is the phagolysosome, a highly acidic organelle,rich in hydrolases, where the internalized microorganisms arekilled and degraded. The importance of phagosomal matura-tion is highlighted by the ability of some intracellular patho-gens to arrest this process (13, 14). By interfering with normalmaturation, several microorganisms like Mycobacterium spe-cies, are capable of surviving for extended periods within im-mature phagosomes of macrophages (15–18).

During the course of maturation, phagosomes progressivelyacquire a variety of proteins that are characteristic of endo-somes and lysosomes (4, 9, 19–21). One of the protein com-plexes inserted into the phagosomal membrane through fusionwith endomembranes, the vacuolar-type ATPase, mediates theacidification of the phagosomal lumen (22–24). Conversely,surface proteins that were internalized during the invaginationof the plasma membrane are gradually removed from the pha-gosome as it matures (9, 25). At least some of these proteinsappear to be recycled back to the plasma membrane (6, 10, 26,27). In this regard, the phagosome has been shown to be acomplete antigen processing compartment, so that MHC-IIzantigen complexes formed therein appear on the plasmamembrane (28).

Despite the importance of vesicular budding in the salvage ofplasmalemmal components, antigen presentation and themaintenance of phagosomal size, little is known about theunderlying mechanisms. In other organelles, such as the Golgicomplex, several fission systems have been characterized, in-cluding the clathrin, COPI, and COPII complexes. Clathrin hasbeen found to associate with the nascent phagosome, althoughits precise role in maturation remains unclear (9, 29). Amor-phous coats, distinct from those generated by clathrin, havealso been found on phagosomes (6). These may be related to theCOP systems of other endomembranes. Of particular interestare recent findings indicating that normal delivery of cargofrom early to late endosomes and lysosomes requires COPI.This was inferred from the observation that microinjection ofantibodies to the b-COP subunit of COPI precluded early tolate endosomal traffic (30). A similar conclusion was reachedusing the ldlF Chinese hamster ovary (CHO)1 cell line, whichhave a temperature-sensitive mutation in e-COP, a subunit of

* This work was supported in part by the Medical Research Council ofCanada, the National Sanatorium Association, and National Institutesof Health Grant AI-22193. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Studentship from the Natural Sciences and Engineer-ing Research Council of Canada.

i International Scholar of the Howard Hughes Medical Institute andcurrent holder of the Pitblado Chair in Cell Biology. To whom corre-spondence should be addressed: Div. of Cell Biology, 555 UniversityAve., Toronto M5G 1X8, Canada. Tel.: 416-813-5727; Fax: 416-813-5028; E-mail: sga@sickkids.on.ca.

1 The abbreviations used are: CHO, Chinese hamster ovary; ARF,adenosine ribosylation factor; DIC, differential interference contrast;FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; LAMP,lysosome-associated membrane protein; RBC, red blood cells; Tf, trans-ferrin; TfR, transferrin receptor; V-ATPase, vacuolar-type H1-pumpingATPase; PAGE, polyacrylamide gel electrophoresis.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 21, Issue of May 26, pp. 15717–15727, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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the COPI complex (31–35). Like wild-type CHO cells, ldlF cellsare normally infected by vesicular stomatitis virus at the per-missive temperature. At the restrictive temperature e-COPbecomes unstable and is rapidly degraded. Under these condi-tions viral infection is completely arrested, indicating the fail-ure of the virus to reach the acidic late endosomes (35). Inaddition, progression of fluid-phase markers like horseradishperoxidase into the late endocytic compartment was at leastpartially blocked (33, 35), as was the case for complexes formedbetween epidermal growth factor and its receptor (35). Theseresults imply that COPI is essential for endosomal progressionand are consistent with the reported effects of brefeldin A onendosomal function and morphology (36–38). Brefeldin A is aspecific inhibitor of GTP exchange factors of the adenosineribosylation factor (ARF) family of GTPases (39) which areessential for COPI function (40, 41).

The involvement of COPI in the endocytic pathway is con-sistent with both the “endosome maturation” and “vesicle shut-tle” models, two competing hypotheses offered to explain howinternalized molecules progress along the endocytic pathway.The vesicle shuttle model proposes that endosomes are stable,long-lived organelles that deliver cargo to the downstream com-partment via vesicular intermediates (42–45). In contrast, thematuration model envisions the gradual modification of endo-cytic compartments by a series of fusion and fission events (19,21, 42, 46–48). While endocytic progression is a subject ofongoing debate, there is little disagreement that phagosomesmature (6). Regardless of the uncertainties regarding endoso-mal progression, there is a striking resemblance between theendocytic and phagocytic pathways. In both cases, vesicles/vacuoles detach from the plasmalemma and acquire sequen-tially Rab5 and Rab7, followed by lysosome-associated mem-brane proteins (or LAMPs) and lysosomal enzymes such ascathepsin (4, 19, 21, 42, 46, 47, 49, 50). Moreover, both systemsundergo a gradual acidification, suggestive of progressive in-sertion of V-ATPases (23, 24, 42, 51, 52). Lastly, as in the caseof endosomes, both ARF and the b-COP subunit of COPI werereported to associate with phagosomal membranes in vitro (6,30, 53).

The similarity between these processes, together with theestablished role of COPI in endosomal progression, promptedus to analyze the role of COPI in phagosomal maturation. Twoapproaches were used: (i) ARF exchange factors were inhibitedin RAW264.7 macrophages using brefeldin A and (ii) the e-COPsubunit of COPI was eliminated using the temperature-sensi-tive mutant ldlF cell line. The latter was derived from CHOcells, which are not professional phagocytes. In order to studyphagosomal maturation in ldlF cells, they were heterologouslytransfected with cDNA encoding the human FcgRIIA receptor.It was shown earlier that expression of Fcg receptors suffices toconfer phagocytic capabilities to nonmyeloid cells such as COSand CHO (54, 55). Such transfectants not only internalizeIgG-opsonized particles, but the resulting phagosomes undergoa maturation process that faithfully recapitulates the matura-tion sequence described in professional phagocytes (56). Com-parison of the rate and extent of maturation when FcgRIIA-transfected ldlF cells were incubated at the permissive orrestrictive temperatures allowed us to assess the role of COPIin vesicular fission from phagosomes.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Dulbecco’s modified Eagle’s medium andfetal bovine serum were from Life Technologies. Bafilomycin, concana-mycin, and G418 were from Calbiochem (La Jolla, CA). Brefeldin A wasobtained from the Upjohn Laboratories (Kalamazoo, MI). Fluoresceinisothiocyanate (FITC), lysine-fixable FITC-dextran Mr 3000, FITC- andrhodamine-conjugated human transferrin (Tf), monensin, nigericin, Or-egon Green-dextran Mr 10,000 and zymosan were from Molecular

Probes (Eugene, OR). Sheep red blood cells (RBC) and rabbit anti-sheepRBC antibodies were from ICN-Cappel. Human IgG and mouse anti-tubulin antibodies were from Sigma. Mouse anti-hamster (UH1) andrat anti-mouse (1D4B) LAMP1 antibodies were from the DevelopmentalStudies Hybridoma Bank, maintained by the University of Iowa and theJohns Hopkins University School of Medicine (Baltimore, MD). Mono-clonal anti-Tf receptor antibody was from Zymed Laboratories Inc. (SanFrancisco, CA). Mouse anti-FcgRIIA monoclonal antibody IV.3 wasfrom Medarex (Annandale, NJ). Monoclonal anti-giantin and rabbitanti-e-COP antibodies were gifts of Drs. H. P. Hauri (University ofBasel Switzerland) and M. Krieger (Massachusetts Institute of Tech-nology, MA), respectively. FITC-conjugated anti-rabbit, anti-human,anti-mouse, and anti-rat, Cy3-conjugated anti-mouse, and HRP-conju-gated anti-mouse and anti-rabbit antibodies were from Jackson Immu-noResearch Laboratories (West Grove, PA).

Cell Culture and Transfection—ldlF cells were the generous gift ofDr. M. Krieger. CHO and RAW264.7 cells were obtained from theAmerican Type Culture Collection (Rockville, MD).

CHO and ldlF cells were transfected with cDNA encoding theFcgRIIA by the calcium phosphate co-precipitation method of Chen andOkayama (57) and stable transfectants were selected with 2 g/literG418. Resistant cells were cloned and screened for FcgRIIA expressionand for the ability to internalize IgG-opsonized particles. The resultingstable lines, called hereafter FcR-CHO and FcR-ldl, were maintained inDulbecco’s modified Eagle’s medium supplemented with 10% fetal bo-vine serum and 2 g/liter G418. RAW264.7 and FcR-CHO cells wereincubated at 37 °C while ldlF and FcR-ldl were maintained at thepermissive temperature of 34 °C. Degradation of e-COP was induced byshifting FcR-ldl cells to the restrictive temperature of 39 °C for theindicated period. All cell lines were cultured under a humidified atmo-sphere with 5% CO2.

Transient transfection of the human transferrin receptor (TfR) intoFcR-ldl cells was accomplished using Fugene 6 (Roche Molecular Bio-chemicals), according to the manufacturer’s instructions. The pCDM8vector encoding human TfR was a kind gift of Dr. J. Bonifacino (Na-tional Institutes of Health, Bethesda, MD).

SDS-PAGE and Immunoblotting—FcR-ldl and FcR-CHO cells weregrown to confluency and incubated at 39 °C for the indicated timepoints. Whole cell extracts were subjected to SDS-PAGE on 12% poly-acrylamide gels under reducing conditions, according to the method ofLaemmli (58). Proteins were transferred onto a polivinylidene difluo-ride membrane (Millipore Corp., Bedford, MA), blocked with 5% milkfor 1 h and incubated with rabbit anti-e-COP at 1:8000 or mouseanti-tubulin at 1:5000 for 1 h, followed by donkey HRP-conjugatedanti-rabbit or anti-mouse antibodies (1:5000) for 1 h. Enhanced chemi-luminescence was used for visualization. Immunoblots were digitallyscanned and the intensity of the immunoreactive bands was quantifiedusing the NIH Image software.

Phagocytosis Assays—Sheep RBC were opsonized with rabbit anti-sheep RBC antibody at 1:50 for 1 h at room temperature and washedthree times with phosphate-buffered saline. Typically '50 RBC wereadded per macrophage or FcR-ldl cell to initiate phagocytosis. To limitthe time of phagocytosis, adherent external RBC were lysed by hypo-tonic shock as described earlier (59). All incubations during the phag-ocytosis and maturation assays were at 34 °C.

To measure intraphagosomal pH, zymosan was labeled with bothOregon Green 514 and FITC as described (56). Labeled zymosan wasthen opsonized with 1 mg/ml human IgG for 1 h at 37 °C and washedthree times. FcR-ldl cells were then allowed to internalize opsonizedzymosan for 1 h before pH measurements were initiated.

Receptor-mediated and Fluid-phase Endocytosis—To label the earlyendocytic compartment, FcR-ldl and RAW264.7 cells were preincubatedin serum-free medium for 1 h and then incubated with 50 mg/ml rho-damine-Tf for 1 h. Membrane-bound Tf was cleared by a 7-min chasebefore initiation of phagocytosis. To examine progression of internalizedaggregated IgG to late endosomes/lysosomes, human IgG at 10 mg/mlwas aggregated by heating at 65 °C for 20 min. Insoluble complexeswere then sedimented and 1 mg/ml soluble aggregated IgG was addedto cells for 30 min. After washing, internalized IgG complexes werechased for 2 h, fixed, and prepared for detection of LAMP1 by immu-nofluorescence. Progression of material taken up by fluid-phase endo-cytosis to the late endosomes/lysosomes was examined by incubatingcells with 250 mg/ml FITC-dextran lysine-fixable, Mr 3000 for 30 minand chasing for 4 h.

Immunofluorescence and Confocal Microscopy—Late endosomes andlysosomes in FcR-ldl cells were identified by labeling with anti-hamsterLAMP1 monoclonal antibody (undiluted) after fixation with methanol.Fixation with 4% paraformaldehyde and methanol permeabilization

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was used instead when simultaneous FITC-dextran labeling and im-munostaining for hamster LAMP1 was necessary. Murine LAMP1 wasdetected with rat anti-LAMP1 at 1:2 dilution after paraformaldehydefixation and methanol permeabilization. FcgRIIA, the Golgi apparatus,and TfR were labeled with monoclonal antibodies to FcgRIIA (1:200),giantin (1:1000), or TfR (1:250), respectively, after fixation with 4%paraformaldehyde and permeabilization with 0.1% Triton. Fluoro-chrome-conjugated secondary anti-human, anti-mouse, anti-rat, andanti-rabbit were all used at 1:1000. Samples were analyzed with a Leicafluorescence microscope (model DMIRB) with a 3100 oil immersionobjective and the appropriate filter set. Cells and phagosomes wereclearly identifiable under DIC optics. Images were digitally acquiredwith a cooled charge-coupled device camera controlled by the Winviewsoftware (Princeton Instruments, Trenton, NJ). Confocal microscopywas performed with a Zeiss LSM 510 laser scanning confocalmicroscope.

Ratiometric Fluorescence Microscopy—For measurement of intraor-ganellar pH, sorting and recycling endosomes were labeled withFITC-Tf as described above and were identified by their peripheral andjuxtanuclear localization, respectively. To measure the lysosomal pH,cells were labeled with 1 mg/ml Oregon Green-dextran Mr 10,000 for1 h, chased for 4 h, and subsequently incubated at the indicated tem-perature overnight before imaging. Phagosomal pH was measured bycovalently labeling zymosan with pH-sensitive probes, as detailedabove.

Resting pH values were obtained in cells bathed in sodium-richmedium (140 mM NaCl, 5 mM glucose, 15 mM Hepes, pH 7.4) at 37 °C.Calibration of fluorescence versus pH was obtained by substituting thesodium-rich buffer with potassium-rich medium (140 mM KCl, 5 mM

glucose, 15 mM Hepes), adjusted to the desired pH with KOH, followedby addition of the cation/H1 ionophores monensin (2 mM) and nigericin(5 mM). Internalized zymosan particles were identified by their insen-sitivity to abrupt changes in extracellular pH and by their responsive-ness to the addition of 10 mM NH4Cl (see Ref. 60 for details). Themicroscope and software set up used for ratio imaging have been de-scribed in detail elsewhere (60, 61).

RESULTS

ldlF Cells Expressing the Human FcgRIIA Receptor ArePhagocytic—The interaction of b-COP and ARF with phagoso-mal membranes in vitro (6) and the role played by COPI in the

progression of the endocytic pathway (30, 33, 35, 53) weresuggestive of a role of COPI in vesicular budding during pha-gosome maturation. We used two different approaches to ana-lyze the involvement of COPI in phagosomal maturation: (i)inhibition of ARF nucleotide exchange factors using brefeldin Ain the professional phagocyte cell line RAW264.7 and (ii) elim-ination of e-COP in the temperature-sensitive mutant ldlFcells.

Because ldlF cells are not normally phagocytic, they weretransfected with the human FcgRIIA receptor gene and stableclones were selected. The selected clones, termed FcR-ldl, ex-pressed the FcgRIIA receptor on their plasma membrane (Fig.1A) and were capable of binding IgG-opsonized RBC (Fig. 1B).More importantly, FcR-ldl cells were able to internalize opso-nized RBC (Fig. 1C), as described earlier for FcgRIIA-trans-fected wild-type CHO cells (56). In fact, the phagocytic effi-ciency of the FcR-ldl clone selected for the subsequentexperiments was greater than reported for FcgRIIA-trans-fected CHO cells (.50 versus ,30%). Phagocytosis of RBC byldlF cells was strictly dependent on opsonization of the parti-cles with IgG and occurred only in cells transfected withFcgRIIA receptors (not illustrated).

FcR-ldl Cells Degrade e-COP at the Restrictive Tempera-ture—We next examined whether FcR-ldl cells retained thetemperature-sensitive mutation in e-COP. The effect of pro-gressively longer incubations at the restrictive temperature(39 °C) on the e-COP content of FcR-ldl cells was analyzed byimmunoblotting. To ensure comparable loading, the sampleswere also probed for tubulin, an abundant protein that shouldbe unaffected by the ldlF mutation. For comparison, wild-typeCHO cells transfected with FcgRIIA (named hereafter FcR-CHO) were also incubated at 39 °C for identical periods. Whenmaintained at the permissive temperature (34 °C) the e-COPcontent of FcR-ldl cells is about 3–4-fold lower than that ofFcR-CHO cells (not shown). In addition, as shown in Fig. 2,

FIG. 1. FcR-ldl cells bind and internalize IgG-opsonized particles. ldlF cells were transfected with the human FcgRIIA receptor and stableclones, termed FcR-ldl were selected. A, the surface expression of FcgRIIA was verified by immunostaining fixed cells with the IV.3 monoclonalantibody. B, IgG-opsonized RBC were added to FcR-ldl cells and incubated on ice for 15 min. Unbound RBC were washed with phosphate-bufferedsaline and images were captured using DIC optics. C, FcR-ldl cells were incubated with IgG-opsonized RBC for 30 min at 34 °C and unbound RBCwere washed away. Externally adherent RBC were lysed by hypotonic shock. Images of live cells were captured using DIC optics. Arrowheads pointto internalized RBC, which resist hypotonic lysis. Scale bar: 10 mm.

FIG. 2. e-COP is degraded in FcR-ldl cells at the restrictive temperature. FcR-CHO and FcR-ldl cells were grown to confluency at 34 °Cand shifted to 39 °C for the indicated times (in hours). Whole cell extracts were then prepared in SDS and aliquots of 40 mg of protein weresubsequently separated by SDS-PAGE and transferred to a polyvinyilidene difluoride membrane. The blot was then incubated with 1:8000 rabbitanti-e-COP and 1:5000 monoclonal anti-tubulin antibodies, followed by 1:5000 HRP-conjugated anti-rabbit and anti-mouse antibodies, respec-tively. Enhanced chemiluminescence was used to detect the immunoreactive bands. Representative of four similar experiments.

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e-COP rapidly diminishes when the mutant cells are warmed to39 °C, while the level of tubulin remains unaffected. By con-trast, e-COP in FcR-CHO cells did not diminish after 6 h at therestrictive temperature (leftmost lane in Fig. 2), as reported forwild-type CHO cells (32). After 6 h at 39 °C, the e-COP contentof FcR-ldl cells is less than 5% of the content in FcR-ldl cells

maintained at the permissive temperature and less than 2% ofthe content of FcR-CHO cells. These findings confirm that thetemperature-sensitive mutation of the parental ldlF cell linewas preserved in our FcR-ldl clonal line. All subsequent exper-iments were performed in cells incubated at 39 °C for 8 h.

In ldlF cells, the loss of e-COP alters the function of the COPI

FIG. 3. Golgi and endosome func-tions are disrupted in COPI-deficientFcR-ldl cells. A-B, FcR-ldl cells treatedat 34 °C (A) or 39 °C for 8 h (B) wereimmunostained with a monoclonal anti-giantin antibody. In COPI-containingcells (A) the Golgi was seen as a densejuxtanuclear cluster, likely a stack of cis-ternae. In COPI-deficient cells (B), theGolgi was dispersed and vesiculated. Cand D, endosome function in FcR-ldl cellsmaintained at 34 °C. The cells were la-beled with 250 mg/ml of lysine-fixableFITC-dextran for 30 min and chased for4 h. Cells were then fixed, permeabilizedwith methanol, and immunostained forLAMP1. C, distribution of LAMP1. D, dis-tribution of FITC-dextran. E and F, endo-some function in cells lacking e-COP.FcR-ldl cells were shifted to 39 °C for 8 hand treated as described for C and D. E,LAMP1; F, FITC-dextran. Insets show im-ages of cells acquired immediately afterthe dextran pulse, demonstrating that pi-nocytosis of dextran persisted in the ab-sence of COPI. Scale bar: 10 mm. Repre-sentative of three similar experiments.

FIG. 4. e-COP deficiency alters thesubcellular distribution of aggre-gated IgG. FcR-ldl cells were preincu-bated at 34 °C (A and B) or at 39 °C (Cand D) and then allowed to internalizeaggregated human IgG for 30 min, fol-lowed by a 2-h chase. After fixation, cellswere immunostained for LAMP1 (A andC) and treated with FITC-conjugated an-ti-human IgG antibodies to reveal the lo-cation of the aggregates (B and D). Rep-resentative of two experiments.

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complex, as judged by alteration of the Golgi morphology andby disruption of endosome function (31, 33, 35). We testedwhether depletion of e-COP had similar functional conse-quences in FcR-ldl cells. Immunofluorescence was used to an-alyze the distribution of giantin, a Golgi resident protein. FcR-ldl cells maintained at 34 °C displayed a typical Golgimorphology, consisting of tightly packed juxtanuclear cisternae(Fig. 3A). Following incubation for 8 h at the restrictive tem-perature, the Golgi marker became dispersed and vesiculated(Fig. 3B), consistent with inactivation of COPI upon loss ofe-COP (31).

Normal progression of endocytic markers from sorting to lateendosomes has also been shown to depend on COPI. Thus,infection of cells with vesicular stomatitis virus, which requiresentry into acidic late endosomes, was inhibited by microinjec-tion of inhibitory antibodies to b-COP (30) and was also im-paired in ldlF cells incubated at restrictive temperatures (35).In the latter, transit of internalized HRP and epidermal growthfactor to late endosomes/lysosomes was similarly defective (33,35). We tested the traffic along the endocytic pathway of FcR-ldl cells by monitoring the fate of fixable FITC-dextran inter-nalized by fluid phase uptake. FcR-ldl cells maintained at34 °C or preincubated for 8 h at 39 °C were allowed to take upthe dextran for 30 min and then chased for 0 or 4 h at therespective temperatures. After fixation, late endosomes andlysosomes were identified by immunostaining for LAMP1. Cellsmaintained at 34 °C accumulated FITC-dextran in LAMP1-positive organelles (Fig. 3, C and D). In sharp contrast, treat-ment at 39 °C significantly reduced the amount of FITC-dex-tran in these organelles (Fig. 3, E and F), although theinhibition was not complete. Pinocytosis was not impaired in

the e-COP-deficient cells, since FITC-dextran was readily de-tectable within the cells immediately after the pulse (Fig. 3,inset). Jointly, these observations indicate that FcR-ldl cellsmaintained the e-COP temperature-sensitive mutation andthat the loss of this subunit is associated with inhibition ofCOPI function.

Traffic of Immune Complexes along the Endocytic Pathway IsAbnormal in the Absence of e-COP—Fcg receptors bound to IgGimmune complexes were shown to be internalized and targetedfor degradation in late endosomes and lysosomes (62, 63). Thisis an essential step for antigen processing and presentation.However, the mechanism by which immune complexes travelalong the endocytic pathway is not known. We examinedwhether COPI is involved in this process using FcR-ldl cells.

FcR-ldl cells treated at the permissive or restrictive temper-atures were allowed to take up aggregated IgG and were sub-sequently chased and stained for LAMP1. As expected, aggre-gated IgG overlapped extensively with LAMP1-positiveorganelles in cells expressing functional COPI (Fig. 4, A and B).These structures were usually small, punctate, and numerous.In the absence of e-COP, IgG complexes were also internalizedand found to accumulate in LAMP1-positive organelles (Fig. 4,C and D). However, significant differences were noted. First,the number of IgG-positive organelles was considerably lowerin COPI-deficient cells. Second, these structures often ap-peared to be larger and mainly perinuclear (cf. Fig. 4, D and B).As a result, only a subset of LAMP1-expressing organellesco-localized with the aggregated IgG, unlike the control cellswhere co-localization was extensive. These results suggest thata sub-set of the LAMP1-containing organelles is inaccessible tothe Fc receptor-ligand complex in cells lacking e-COP. We

FIG. 5. The Golgi and endosomefunctions are disrupted by brefeldinA in RAW264.7 macrophages. A and B,RAW264.7 cells were incubated in the ab-sence (A) or presence (B) of 100 mM brefel-din A for 45 min and fixed. The Golgi wasdetected with monoclonal anti-giantin an-tibodies as described in the legend to Fig.3. C-F, cells were labeled with 250 mg/mllysine-fixable FITC-dextran for 30 minand chased for 4 h. Cells were then fixedand immunostained for LAMP1. The dis-tribution of LAMP1 (C and E) and FITC-dextran (D and F) was visualized by con-focal microscopy. C and D, control cells. Eand F, brefeldin-treated cells. Represent-ative of three experiments.

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conclude that COPI plays a role in the normal traffic of immunecomplexes along the endocytic pathway.

Brefeldin A Disrupts the Golgi and Impairs Endosome Func-tion in RAW264.7 Macrophages—To study the role of COPI inphagosome maturation in cells of myeloid origin that are in-trinsically phagocytic, we used brefeldin A. Brefeldin A is afungal metabolite widely used to disrupt COPI function. Itassociates with and blocks the activity of nucleotide exchangefactors that regulate ARF (39, 40). Because brefeldin A is notuniversally effective in all cell types (36, 64) we tested itseffectiveness in RAW264.7 cells.

As above, the morphology of the Golgi complex in untreatedRAW264.7 cells was compact and juxtanuclear (Fig. 5A). Fol-lowing treatment with brefeldin, however, the Golgi markergiantin became dispersed and punctate (Fig. 5B). Next, endo-some function was examined in RAW264.7 macrophages

treated with brefeldin A. As observed for FcR-ldl cells, FITC-dextran efficiently accumulated in LAMP1-positive organellesin untreated RAW264.7 cells (Fig. 5, C and D). However, treat-ment with brefeldin significantly reduced, but did not entirelypreclude, the accumulation of FITC-dextran in these organelles(Fig. 5, E and F). Analysis of the cells immediately after thepulse revealed that pinocytic uptake of FITC-dextran was notgreatly affected, as described above for FcR-ldl cells. Thesedata are consistent with the inhibition of HRP progression tolate endosomes/lysosomes in brefeldin A-treated NRK and ratyolk epithelial cells (37, 51), although discrepant results havebeen reported in other cell types (38). Our results imply thatbrefeldin is an effective tool to study the role of COPI inmacrophages.

Phagocytosis in COPI-deficient Cells—Analysis of the role ofCOPI in phagosomal maturation requires that particle inter-nalization be preserved in the absence of functional COPI.Zhang et al. (65) described earlier that phagocytosis persistedin RAW264.7 cells treated acutely with brefeldin A (65). We

FIG. 6. TfR recycling from phagosomes in FcR-ldl cells. FcR-ldlcells were allowed to internalize IgG-opsonized RBC for 15 min. Cellswere then fixed immediately or following a 15- or 30-min chase. TfR wasidentified with a monoclonal anti-TfR antibody followed by Cy3-conju-gated anti-mouse antibody. Internalized RBC were identified with DICoptics or with FITC-conjugated anti-rabbit antibody that bound to theopsonizing rabbit IgG. A, TfR staining in cells fixed immediately afterthe phagocytic pulse. B, corresponding DIC image of opsonized RBC. C,TfR staining in cells fixed 30 min after the phagocytic pulse. D, corre-sponding stained RBC. Arrowheads point to RBC. Bar represents 10mm. E, course of disappearance of TfR from phagosomes. FcR-ldl cellsmaintained at 34 °C (solid bars) or 39 °C (open bars) were treated asdescribed above. The cells were analyzed microscopically and phago-somes lined by observable TfR were defined as TfR-positive. Resultswere collected from three different experiments with 150 phagosomesper condition. Data are mean 6 S.E. At all time points, the differencebetween e-COP containing and deficient cells was statistically signifi-cant (p , 0.05, Student’s t test).

FIG. 7. TfR recycling from phagosomes in RAW264.7 macro-phages: effect of brefeldin. Macrophages were allowed to internalizeIgG-opsonized RBC for 15 min. Cells were then fixed immediately orfollowing a 15 or 30 min chase without RBC at 34 °C. TfR and inter-nalized RBC were identified as described in Fig. 5, but using confocalmicroscopy. A, TfR staining in cells fixed immediately after the phag-ocytic pulse. B, corresponding staining of opsonized RBC. C, TfR stain-ing in cells fixed 30 min after the phagocytic pulse. D, correspondingRBC. Arrowheads point to RBC. Bar represents 10 mm. E, course ofdisappearance of TfR from phagosomes. Control (solid bars) or brefel-din-treated cells (open bars) were processed as described above. Resultswere collected from three different experiments with 150 phagosomesper condition. Data are mean 6 S.E. The difference between control andbrefeldin-treated samples was statistically significant at all time peri-ods tested (p , 0.05 using the paired Student’s t test).

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were able to confirm these observations in RAW264.7 cellsincubated with 100 mM brefeldin for 45 min, conditions shownabove to disrupt the Golgi and to interfere with endosomaltraffic. In addition, we found that FcR-ldl cells that had nodetectable e-COP following incubation at 39 °C for 8 h werenevertheless able to internalize IgG-opsonized RBC, althoughthe phagocytic efficiency was only '50% relative to cells main-tained at the permissive temperature. This partial inhibition ofphagocytosis was specific to the disappearance of e-COP andwas not a deleterious effect of heat shock, since FcR-CHO cellsincubated at 34 and 39 °C had similar phagocytic efficiencies.The mechanism underlying the inhibition of phagocytosis ine-COP-deficient cells will be described in detail elsewhere.2

Nevertheless, the persistence of phagocytosis in the absence ofe-COP enabled us to study the role of COPI in phagosomalmaturation.

Role of COPI in Removal of TfR from the Phagosomal Mem-brane—Transferrin receptors are incorporated into the phago-somal membrane during particle ingestion and are also in-serted by fusion with early endosomes (15, 56, 66). However,the residence time of TfR in the phagosome is limited, as theyare removed by vesicular budding within 10–20 min of phago-some closure (6, 25, 56, 67). The possible role of COPI in suchbudding was analyzed by immunofluorescence.

Phagocytosis was initiated by exposing e-COP-containing ordepleted FcR-ldl cells to IgG-opsonized RBC for 15 min at34 °C. After this phagocytic pulse, cells were either fixed im-mediately or chased (i.e. incubated in the absence of RBC) for15 or 30 min also at 34 °C and the distribution of TfR deter-mined by immunostaining. As anticipated, TfR were detectedon some phagosomes immediately after the phagocytic pulse(Fig. 6, A and B; note that even at this early time approxi-mately 60% of the phagosomes had lost or never acquired TfR).However, after a 30-min chase, practically all phagosomes hadno detectable TfR (Fig. 6, C and D). A comparison of the ratesof disappearance of TfR from the phagosomes is presented inFig. 6E, which summarizes the results of three experiments,where a total of 450 phagosomes were counted. At all timesstudied, COPI-expressing FcR-ldl cells had more effectivelyremoved TfR receptors from their phagosomes than theirCOPI-deficient counterparts. The differences in the number ofTfR-positive phagosomes were found to be statistically signifi-cant 15, 30, and 45 min after initiation of phagocytosis (i.e. 0,15, or 30 min of chase, respectively, in Fig. 6). These findingssuggest that COPI plays a role in the early stages of phagoso-mal maturation.

Similar conclusions were drawn when comparing the fate ofTfR in control and brefeldin A-treated RAW264.7 macro-phages. As illustrated in Fig. 7, A and B, TfR was enriched inphagosomes immediately after phagocytosis was completed. Asreported earlier (15, 56, 66), the receptors were rapidly clearedfrom the phagosomes of otherwise untreated cells (Fig. 7, C andD). As in the case of COPI mutants, the rate of disappearanceof TfR receptors from the phagosome was noticeably slowerwhen brefeldin was present (Fig. 7E). Even at the earliest timemeasured (0 min chase after the 15-min phagocytic pulse) thefraction of TfR-positive phagosomes was significantly greaterin brefeldin-treated samples than in controls (p , 0.05). Be-cause only partial inhibition was observed when COPI wasgenetically ablated or inhibited pharmacologically, we concludethat TfR removal from phagosomes is mediated by at least twodistinct mechanisms: one that is COPI-dependent and one ormore COPI-independent processes.

Role of COPI in Removal of Fcg Receptors from the Phagoso-mal Membrane—After signaling particle internalization, Fcgreceptors (FcgR) are no longer required on the phagosomalmembrane and are eventually cleared (9, 63). Because little isknown about the mechanism(s) underlying FcgR removal fromphagosomes, we investigated the possible role of COPI in thisprocess. The occurrence of this phenomenon, which has beendescribed in professional phagocytes, was initially verified inFcR-ldl cells grown under permissive conditions. Fig. 8, A-D,shows that, while FcgRIIA receptors are highly concentrated innascent and early phagosomes, they gradually disappear overtime. After a 60-min chase, Fcg receptors were detectable inonly '40% of the phagosomes. In contrast to the results ob-tained with TfR, the rate of clearance of Fcg receptors wasunaffected when e-COP was eliminated by preincubating thecells at the restrictive temperature (Fig. 8E). Therefore,FcgRIIA receptors are removed from phagosomes in a COPI-independent manner.

2 D. J. Hackam Botelho, R. J., C. Sjolin, D. D. Rotstein, J. M.Robinson, A. D. Schreiber, and S. Grinstein, manuscript in preparation.

FIG. 8. FcgRIIA removal from phagosomes in FcR-ldl cells.FcR-ldl cells were allowed to internalize IgG-opsonized RBC for 15 min.Cells were then fixed immediately, or following a 15- or 30-min chase,and immunostained with a monoclonal anti-human FcgRIIA antibody.A, FcgRIIA staining in cells fixed immediately after the phagocyticpulse. B, corresponding bright field image. C, FcgRIIA staining in cellsfixed 60 min after the phagocytic pulse. D, corresponding bright fieldimage. Arrowheads point to RBC. Bar represents 10 mm. E, course ofdisappearance of FcgRIIA from phagosomes. FcR-ldl cells maintainedat 34 °C (solid bars) or 39 °C (open bars) were treated as describedabove. The cells were analyzed microscopically and phagosomes linedby observable FcgRIIA were defined as FcgRIIA-positive. Results werecollected from three different experiments with 150 phagosomes percondition. Data are mean 6 S.E. No statistically significant differencebetween e-COP containing and deficient cells was found at any time(p . 0.05, Student’s t test).

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Fusion of Early Endosomes with Phagosomes in the Absenceof COPI—Phagosomes acquire proteins which are essential fortheir microbicidal function, such as cathepsins and V-ATPases,by fusing with endosomes and lysosomes. The sequential fusionsteps undergone by phagosomes closely resemble the process ofendosomal maturation (42, 46, 50). Because the latter wasshown to be arrested by deletion of e-COP, we tested whetherphagosomal maturation was similarly dependent on COPI.

We initially used FcR-ldl cells to assess the role of e-COP. Tomonitor the fusion of early endosomes with the phagosome,cells were pre-loaded with rhodamine-Tf for 1 h, followed by achase period intended to clear any Tf bound to the plasmamembrane. The cells were next allowed to internalize opso-nized RBC and, after arresting the reaction, the subcellulardistribution of Tf was analyzed. As reported for FcR-CHO (56),FcR-ldl maintained at 34 °C undergo rapid and efficient fusionwith Tf-containing endosomes (Fig. 9, A and B). The distribu-tion of rhodamine-Tf on the phagosomal membrane was oftenuneven, perhaps reflecting asynchrony in the onset of phago-cytosis. When FcR-ldl cells were pretreated at 39 °C to elimi-nate e-COP, phagosomes were nevertheless able to acquire Tf,indicating that phagosome-early endosome fusion proceeded inthe absence of COPI (Fig. 9, C and D).

Similar observations were made in control and brefeldinA-treated RAW264.7 macrophages. Tf loaded into early endo-somes readily reached the phagosome, whether COPI was func-tional or had been disassembled by pretreatment with brefel-din (data not shown).

Fusion of Late Endosomes/Lysosomes with Phagosomes inthe Absence of COPI—The fusion of phagosomes with late en-dosomes and/or lysosomes was monitored using LAMP1 as a

marker. Unlike the rapid and transient acquisition of Tf,LAMP1 reaches the phagosomes in a slower and more sus-tained fashion. In FcR-ldl cells maintained at the permissivetemperature, the number of phagosomes that were immunore-active toward LAMP1 increased progressively throughout the30-min chase period (Fig. 10). Ablation of e-COP by preincuba-tion at 39 °C had only a small, statistically insignificant effecton the rate and extent of LAMP1 fusion with phagosomes (Fig.10E).

That COPI was not essential for fusion between phagosomesand late endosomes/lysosomes was confirmed in RAW264.7cells using brefeldin. Pretreatment of the cells with this inhib-itor had no effect on the extent of LAMP1 fusion with phago-somes (not illustrated). We conclude that COPI is not requiredfor the intermediate stages of phagosomal maturation.

Acidification of COPI-deficient Phagosomes and Endo-somes—Acquisition of LAMP1 is not a definitive indicator ofthe completion of phagosomal maturation. Thus, phagosomescontaining live mycobacteria do not fuse with lysosomes nor dothey acidify fully, yet acquire LAMP1 (15, 16, 61). To ensurethat bona fide phagolysosomes are formed in the absence ofe-COP we measured phagosomal pH in FcR-ldl cells. Followingincubation at either the permissive or restrictive temperature,FcR-ldl cells were allowed to internalize zymosan particlescovalently labeled with pH-sensitive fluorescent probes and thephagosomal pH was measured by ratio microfluorimetry. Asdescribed earlier, internalized zymosan particles were identi-fied by their responsiveness to addition of NH4Cl and by theirinsensitivity to abrupt changes in extracellular pH (not shown).In cells with functional COPI the phagosomal pH averaged4.2 6 0.2 (mean 6 S.E. of 23 determinations; Table I). Follow-

FIG. 9. Fusion of phagosomes with early endosomes in FcR-ldl cells. The early endosomal compartment was labeled by preincubation ofserum-starved cells with rhodamine-Tf for 1 h. Plasma membrane-bound Tf was chased for 7 min and the cells were allowed to internalizeIgG-opsonized RBC for 15 min. After hypotonic shock to remove adherent external RBC, the cells were visualized by fluorescence and DICmicroscopy. A, Tf distribution in FcR-ldl cells maintained at 34 °C. B, corresponding DIC image. C, Tf distribution in FcR-ldl cells preincubatedat 39 °C to degrade e-COP. D, corresponding DIC image. Bar represents 10 mm. Representative of three experiments.

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ing degradation of e-COP, the phagosomal pH reached 4.5 6 0.2(n 5 14), a level that is not significantly different from thecontrol (p . 0.05). In both instances the acidification wasshown to be V-ATPase dependent, since inhibition of these H1

pumps with bafilomycin or concanamycin neutralized the pha-gosomal pH (not shown). Therefore, it appears that bona fidephagolysosomes, capable of acidification and of acquiringLAMP1, can form in the absence of e-COP in FcR-ldl cells.

Endosomal and lysosomal pH had been assessed qualita-tively in e-COP-deficient cells by Daro et al. (35) and Gu et al.(33). We used the more quantitative ratiometric microfluorim-etry method to confirm their observations. Endosomal pH wasassessed using fluoresceinated Tf. As summarized in Table I,peripheral endosomes were more acidic than those clustered inthe juxtanuclear recycling compartment. However, no differ-ences were noted for either compartment between cells incu-bated at the permissive or restrictive temperatures, in accord-ance with the earlier results (33, 35).

The pH of lysosomes was also measured. To deliver a pH-sensitive probe to this compartment, the cells were allowed tointernalize labeled dextran prior to the temperature shift, topermit traffic along the endocytic pathway. After subsequentincubation at either 34 or 39 °C, the pH was measured asabove, and found to be similarly acidic in both cases (Table I).The very acidic pH attained by phagosomes, which is similar tothat of lysosomes, is consistent with the notion that V-ATPasesare delivered upon phago-lysosomal fusion in e-COP-deficientcells.

DISCUSSION

While removal of components of the phagosomal membraneis known to occur during the course of maturation, no specificmechanisms of vesicular budding have been identified to me-diate this event. Nonetheless, the striking parallels betweenthe processes of phagosomal and endosomal maturation sug-gested that similar mechanisms might be involved. Of partic-ular interest was the COPI complex, which was recently foundto be essential for normal progression of endosomes (30, 33, 35,53). The possibility that COPI may participate in phagosomematuration was reinforced by the observation that both b-COPand ARF associate with phagosomal membranes (6). We there-fore used a temperature-sensitive COPI mutant cell line, andalso tested the effects of brefeldin A in professional phagocytes.These models are complementary, rather than redundant.Brefeldin inhibits COPI by interfering with the function ofguanine nucleotide exchange factors for the ARF family ofGTPases (39). Since there are a multitude of ARF isoforms andof the corresponding exchange factors, the specificity of brefel-din A for COPI is not absolute (68). In fact, association of theadaptor proteins AP-3 and AP-4 with membranes was shown tobe disrupted by brefeldin A (69–71). Furthermore, some celltypes resist disruption of the Golgi complex after treatmentwith brefeldin (36, 64), suggesting that in these systems COPIis controlled by brefeldin-insensitive ARF exchange factors.

The specificity of the FcR-ldl model is in principle superior.

To our knowledge, only e-COP is directly affected by the shift tothe restrictive temperature in these cells. However, the com-paratively long time required for the total disappearance ofe-COP (.6 h) may have secondary consequences. Indeed, theefficiency of phagocytosis was significantly decreased after 6–8h at 39 °C in FcR-ldl, but not in wild-type FcR-CHO cells (notillustrated). However, the remaining phagocytosis ('50% ofthe control) sufficed for analysis of maturation. Last, althoughphagosomal formation and maturation in the FcR-transfectedldlF cells seem to parallel the events reported in professionalphagocytes, we cannot a priori disregard the possibility thatdifferent budding mechanisms were involved in professionaland engineered phagocytes.

Despite the individual limitations of the two experimentalmodels, the results obtained regarding the role of COPI inphagosomal maturation are internally consistent. First, weconfirmed earlier findings that, at the time points studied,

FIG. 10. Fusion of phagosomes with late endosomes and/orlysosomes in FcR-ldl cells. FcR-ldl cells were allowed to internalizeIgG-opsonized RBC for 1 h, treated hypotonically to remove externalRBC and fixed. The cells were then immunostained with monoclonalanti-hamster LAMP1 antibody. A, LAMP1 staining in cells maintainedat 34 °C. B, corresponding DIC image. C, LAMP1 staining in cellstreated at 39 °C degrade e-COP. D, corresponding DIC image. Bar 5 10mm. E, course of LAMP1 acquisition by phagosomes. FcR-ldl cells main-tained at 34 °C (solid bars) or 39 °C (open bars) were allowed to inter-nalize RBC for 15 min and either fixed immediately or chased for theindicated times. The cells were analyzed microscopically and phago-somes lined by observable LAMP1 were defined as LAMP1-positive.Results were collected from three different experiments with 100 pha-gosomes per condition. Data are mean 6 S.E. No statistically signifi-cant difference between e-COP containing and deficient cells was foundat any time (p . 0.05, Student’s t test).

TABLE ILuminal pH of peripheral and juxtanuclear endosomes, lysosomes,

and phagosomesThe following abbreviations are used in the table: PE and JE, periph-

eral and juxtanuclear endosomes; Ly, lysosomes; Ph, phagosomes. Thedata presented are the mean 6 S.E. of n $ 12 experiments. For eachexperiment, the pH of multiple organelles in one or more cells wasmeasured and averaged.

PE JE Ly Ph

34 °C 6.2 6 0.2 6.6 6 0.2 4.9 6 0.4 4.2 6 0.239 °C 6.4 6 0.2 6.7 6 0.1 4.7 6 0.2 4.5 6 0.2

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COPI is not essential for particle internalization (65). Second,unlike endosomal progression, we observed that phagosomalmaturation proceeded to completion in the absence of e-COPand in the presence of brefeldin. It is noteworthy that endoso-mal maturation is not equally sensitive to brefeldin in allsystems tested (cf. Refs. 37 and 38) and that progression ofviruses or molecules internalized via fluid-phase or receptor-mediated endocytosis is not equally affected by deletion ofe-COP (35). The alternate pathways that underlie these differ-ences may play a dominant role in phagosome maturation.

While phagosome maturation proceeds to completion in theabsence of functional COPI, as judged by the acquisition ofLAMP1 and by the attainment of a very acidic pH, the traffic ofsome components is altered. Specifically, both experimentalsystems indicated that budding of TfR from the phagosome isaffected by impairment of COPI function. It is important tonote, however, that only partial inhibition of TfR recycling wasobserved in e-COP deficient or brefeldin-treated cells, implyingthat at least one other COPI-independent pathway can be usedfor membrane budding off phagosomes. The possible role ofclathrin-dependent vesicle formation in phagosome maturationis currently under investigation. Alternatively, it is conceivablethat protein transfer between phagosomes and the endocyticcompartment may occur by “kiss-and-run” (4). In this model,phagosomes and endocytic vacuoles do not coalesce into oneorganelle. Rather, membranes and luminal content are ex-changed between the two compartments by a momentary fu-sion followed by fission.

Not all receptors are cleared from the phagosome via identi-cal pathways. FcgRIIA remains on the phagosomal membraneslong after TfR have pinched off and, more importantly, thegradual disappearance of FcgRIIA is unaffected by inactivationof COPI. The fate of phagosomal Fcg receptors remains un-clear: they may be recycled to the plasma membrane or trans-ferred to lysosomes, as found for Fc receptors cross-linked withspecific antibodies (62, 63), or they may be degraded in situ inthe phagosome. Regardless of the specific path taken byFcgRIIA, COPI appears not to be required for its slow elimina-tion from the phagosome.

As mentioned in the Introduction, the process responsible forthe progression of molecules internalized by endocytosis is thesubject of ongoing debate. Some authors believe that endo-somes mature in a manner analogous to that of phagosomes,i.e. the early endosome is felt to be gradually converted into alate endosome and eventually a lysosome by remodeling of itsmembrane and contents that results from multiple fusion andfission events (19, 21, 42, 46–48). Others feel that the early andlate endosomes as well as the lysosomes are stationary compo-nents that deliver the internalized material from one to thenext by means of carrier vesicles (42–45). The formation ofsuch carrier vesicles was proposed to depend on COPI, therebyaccounting for the arrest of progression in the ldlF cells (33,53). In the case of phagosomes, the predominance of matura-tion as opposed to carrier vesicle-mediated progression, mayexplain their relative insensitivity to COPI and their differen-tial behavior compared with endosomes.

Acknowledgments—We are deeply indebted to Dr. M. Krieger (MIT,Boston, MA) for providing the ldlF cell line and the rabbit anti-e-COPantibody. We also thank Dr. J. Bonifacino (National Institutes ofHealth, Bethesda, MD) for providing the mammalian expression vectorcontaining the human transferrin receptor and Dr. H. P. Hauri (Uni-versity of Basel, Switzerland) for the generous gift of antibodies togiantin.

REFERENCES

1. Kwiatkowska, K., and Sobota, A. (1999) Bioessays 21, 422–4312. Silverstein, S. C., Greenberg, S., Di Virgilio, F., and Steinberg, T. H. (1989) in

Fundamental Immunology (Paul, W. E., ed) pp. 703–719, Raven Press, Ltd.,New York

3. Swanson, J. A., and Baer, S. C. (1995) Trends Cell Biol. 5, 89–934. Desjardins, M., Huber, L. A., Parton, R. G., and Griffiths, G. (1994) J. Cell Biol.

124, 677–6885. Desjardins, M., Nzala, N. N., Corsini, R., and Rondeau, C. (1997) J. Cell Sci.

110, 2303–23146. Beron, W., Alvarez-Dominguez, C., Mayorga, L., and Stahl, P. D. (1995) Trends

Cell Biol. 5, 100–1047. Lang, T., de Chastellier, C., Ryter, A., and Thilo, L. (1988) Eur. J. Cell Biol. 46,

39–508. Mayorga, L. S., Bertini, F., and Stahl, P. D. (1991) J. Biol. Chem. 266,

6511–65179. Pitt, A., Mayorga, L. S., Stahl, P. D., and Schwartz, A. L. (1992) J. Clin. Invest.

90, 1978–198310. Ryter, A. (1985) Comp. Immunol. Microbiol. Infect. Dis. 8, 119–13311. Leung, K. P., and Allen, R. D. (1982) Eur. J. Cell Biol. 29, 1–1212. de Chastellier, C., and Thilo, L. (1997) Eur. J. Cell Biol. 74, 49–6213. Haas, A. (1998) Mol. Membr. Biol. 15, 103–12114. Meresse, S., Steele-Mortimer, O., Moreno, E., Desjardins, M., Finlay, B., and

Gorvel, J. P. (1999) Nature Cell Biol. 1, E183–E18815. Clemens, D. L., and Horwitz, M. A. (1995) J. Exp. Med. 181, 257–27016. Clemens, D. L., and Horwitz, M. A. (1996) J. Exp. Med. 184, 1349–135517. Deretic, V., Via, L. E., Fratti, R. A., and Deretic, D. (1997) Electrophoresis 18,

2542–254718. Via, L. E., Deretic, D., Ulmer, R. J., Hibler, N. S., Huber, L. A., and Deretic, V.

(1997) J. Biol. Chem. 272, 13326–1333119. Dunn, K. W., and Maxfield, F. R. (1992) J. Cell Biol. 117, 301–31020. Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A., Grif-

fiths, G., and Huber, L. A. (1994) J. Biol. Chem. 269, 32194–3220021. Ward, D. M., Perou, C. M., Lloyd, M., and Kaplan, J. (1995) J. Cell Biol. 129,

1229–124022. Geisow, M. J., D’Arcy Hart, P., and Young, M. R. (1981) J. Cell Biol. 89,

645–65223. Lukacs, G. L., Rotstein, O. D., and Grinstein, S. (1990) J. Biol. Chem. 265,

21099–2110724. Lukacs, G. L., Rotstein, O. D., and Grinstein, S. (1991) J. Biol. Chem. 266,

24540–2454825. Pitt, A., Mayorga, L. S., Schwartz, A. L., and Stahl, P. D. (1992) J. Biol. Chem.

267, 126–13226. Muller, W. A., Steinman, R. M., and Cohn, Z. A. (1980) J. Cell Biol. 86,

304–31427. Muller, W. A., Steinman, R. M., and Cohn, Z. A. (1983) J. Cell Biol. 96, 29–3628. Ramachandra, L., Song, R., and Harding, C. V. (1999) J. Immunol. 162,

3263–327229. Aggeler, J., and Werb, Z. (1982) J. Cell Biol. 94, 613–62330. Whitney, J. A., Gomez, M., Sheff, D., Kreis, T. E., and Mellman, I. (1995) Cell

83, 703–71331. Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213–122432. Guo, Q., Penman, M., Trigatti, B. L., and Krieger, M. (1996) J. Biol. Chem. 271,

11191–1119633. Gu, F., Aniento, F., Parton, R. G., and Gruenberg, J. (1997) J. Cell Biol. 139,

1183–119534. Hobbie, L., Fisher, A. S., Lee, S., Flint, A., and Krieger, M. (1994) J. Biol.

Chem. 269, 20958–2097035. Daro, E., Sheff, D., Gomez, M., Kreis, T., and Mellman, I. (1997) J. Cell Biol.

139, 1747–175936. Hunziker, W., Whitney, J. A., and Mellman, I. (1991) Cell 67, 617–62737. Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L., and

Klausner, R. D. (1991) Cell 67, 601–61638. Wood, S. A., and Brown, W. J. (1992) J. Cell Biol. 119, 273–28539. Helms, J. B., and Rothman, J. E. (1992) Nature 360, 352–35440. Donaldson, J. G., Cassel, D., Kahn, R. A., and Klausner, R. D. (1992) Proc.

Natl. Acad. Sci. U. S. A. 89, 6408–641241. Palmer, D. J., Helms, J. B., Beckers, C. J., Orci, L., and Rothman, J. E. (1993)

J. Biol. Chem. 268, 12083–1208942. Mukherjee, S., Ghosh, R. N., and Maxfield, F. R. (1997) Physiol. Rev. 77,

759–80343. Gruenberg, J., Griffiths, G., and Howell, K. E. (1989) J. Cell Biol. 108,

1301–131644. Griffiths, G., and Gruenberg, J. (1991) Trends Cell Biol. 1, 5–945. Clague, M. J., Urbe, S., Aniento, F., and Gruenberg, J. (1994) J. Biol. Chem.

269, 21–2446. Thilo, L., Stroud, E., and Haylett, T. (1995) J. Cell Sci. 108, 1791–180347. Futter, C. E., Pearse, A., Hewlett, L. J., and Hopkins, C. R. (1996) J. Cell Biol.

132, 1011–102348. Murphy, R. F. (1991) Trends Cell Biol. 1, 72–8249. Roederer, M., Barry, J. R., Wilson, R. B., and Murphy, R. F. (1990) Eur. J. Cell

Biol. 51, 229–23450. van Deurs, B., Holm, P. K., and Sandvig, K. (1996) Eur. J. Cell Biol. 69,

343–35051. Ichimura, T., Hatae, T., and Ishida, T. (1997) Eur. J. Cell Biol. 74, 41–4852. Overly, C. C., Lee, K. D., Berthiaume, E., and Hollenbeck, P. J. (1995) Proc.

Natl. Acad. Sci. U. S. A. 92, 3156–316053. Aniento, F., Gu, F., Parton, R. G., and Gruenberg, J. (1996) J. Cell Biol. 133,

29–4154. Indik, Z. K., Park, J. G., Hunter, S., Mantaring, M., and Schreiber, A. D. (1995)

Immunol. Lett. 44, 133–13855. Nagarajan, S., Chesla, S., Cobern, L., Anderson, P., Zhu, C., and Selvaraj, P.

(1995) J. Biol. Chem. 270, 25762–2577056. Downey, G., Botelho, R. J., Butler, J. R., Moltyaner, Y., Chien, P., Schreiber,

A. D., and Grinstein, S. (1999) J. Biol. Chem. 274, 28436–2844457. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745–275258. Laemmli, U. K. (1970) Nature 227, 680–68559. Hackam, D. J., Rotstein, O. D., Sjolin, C., Schreiber, A. D., Trimble, W. S., and

COPI and Phagosome Maturation15726

by guest on May 1, 2020

http://ww

w.jbc.org/

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nloaded from

Grinstein, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11691–1169660. Hackam, D. J., Rotstein, O. D., Zhang, W., Gruenheid, S., Gros, P., and

Grinstein, S. (1998) J. Exp. Med. 188, 351–36461. Hackam, D. J., Rotstein, O. D., Zhang, W. J., Demaurex, N., Woodside, M.,

Tsai, O., and Grinstein, S. (1997) J. Biol. Chem. 272, 29810–2982062. Mellman, I., and Plutner, H. (1984) J. Cell Biol. 98, 1170–117763. Mellman, I. S., Plutner, H., Steinman, R. M., Unkeless, J. C., and Cohn, Z. A.

(1983) J. Cell Biol. 96, 887–89564. Ktistakis, N. T., Roth, M. G., and Bloom, G. S. (1991) J. Cell Biol. 113,

1009–102365. Zhang, Q., Cox, D., Tseng, C. C., Donaldson, J. G., and Greenberg, S. (1998)

J. Biol. Chem. 273, 19977–1998166. Alvarez-Dominguez, C., Roberts, R., and Stahl, P. D. (1997) J. Cell Sci. 110,

731–74367. Muller, W. A., Steinman, R. M., and Cohn, Z. A. (1980) J. Cell Biol. 86, 292–30368. Moss, J., and Vaughan, M. (1998) J. Biol. Chem. 273, 21431–2143469. Ooi, C. E., Dell’Angelica, E. C., and Bonifacino, J. S. (1998) J. Cell Biol. 142,

391–40270. Dell’Angelica, E. C., Mullins, C., and Bonifacino, J. S. (1999) J. Biol. Chem.

274, 7278- 728571. Hirst, J., Bright, N. A., Rous, B., and Robinson, M. S. (1999) Mol. Biol. Cell 10,

2787- 2802

COPI and Phagosome Maturation 15727

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Roberto J. Botelho, David J. Hackam, Alan D. Schreiber and Sergio GrinsteinRole of COPI in Phagosome Maturation

doi: 10.1074/jbc.M910068199 originally published online March 15, 20002000, 275:15717-15727.J. Biol. Chem. 

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