HepG2 Cells - The Journal of Biological Chemistry

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Mar 19, 1993 - ldlCp was generously provided by Steve Podos and Dr. M. Krieger ...... Yamashiro, C. T., Kane, P. M., Wolczyk, D. F., Preston, R. A., and ...
THEJOURNAL OF BIOLO~ICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 26,Issue of September 6,pp. 19092-19100,1993 Pri‘nted in U.S.A.

Involvement of the Vacuolar H+-ATPasesin the Secretory Pathway of HepG2 Cells* (Received for publication, March 19,1993,and in revised form, May 27,1993)

Mamadi YillaSQ, Agnes TanlQl,Kouichi Itoll, Kiyoshi Miwall, and Hidde L. PloeghQ** From the &Centerfor Cancer Research. of Bwbgy, Massachusetts Institute of Techmbgy, ,Demrtment . Cambridgi Massachusetts 02139

The macrolide antibiotic concanamycin B is a highly the vacuolar H+-ATPases (V-ATPases)’in particular, in sortselective inhibitor (IC6,, = 5 nM) of the H+-ATPases of ing of soluble proteins to the vacuole, the counterpart of the the vacuolar system. We have examined theeffects of mammalian lysosome, and in sorting along the vacuolar sysconcanamycinB on the constitutivesecretory pathway tem (Yamashiro et al., 1990; Klionsky et al., 1992). Several of the human hepatoma cell line, HepG2. In cells ex- studies have suggested a role for acidified compartments in p d to 10 n M concanamycin B, transport from the membrane trafficking in mammalian cells (reviewed in Mellendoplasmic reticulum to the Golgi occurs at normal man et al., 1986; Anderson and Orci, 1988; Laurie and h b ratee, as determined by pulse-chase analysis of endo- bins, 1991). Acidotropic agents such as chloroquine, primaglycosidase H-sensitive productinconjunction with quine and ammonium chloride, and ionophores such as mosubcellularfractionationexperiments.However, in- nensin havebeencommonlyused to study pH regulation tra-Golgi trafficking orGolgi to plasmamembrane (Uchida et al., 1980; Strous et al., 1985; Mellmanet al., 1986). delivery is significantly impaired. A delay in the onset Although these agents have found widespread use in manipof secretion of themajor secretory proteins, albumin, ulating the pH of intracellular acidicorganelles, the high al-antitrypsin and transferrinis observed. Processing concentrations required to achieve desiredeffects, along with of N-linked glycans by sialyltransferases is inhibited, the secondary nonspecific manifestations of these compounds, resulting in secreted glycoproteins which are modified have limited the scope of their usefulness (Mellman et al., less extensively. In view of the acidicpH of the trum- 1986). Similarly, ionophores such as monensin can dissipate Golgi and the trum-Golgi network, thesestudies sug- ionic gradients across membranes, but their selectivity is not gest that acidification by vacuolar ATPases is critical absolute and gross morphological alterations, particularly of to achieving timely secretion and correct N-linked gly- the Golgi apparatus, occur (Tartakoff, 1983; Mellman et al., can modifications of proteins which follow the consti- 1986). Macrolide antibiotics that selectively block the function of distinct types of H+-ATPases have created new postutive secretory pathway. sibilities for exploring the role of the acidic pH of the transGolgi region. This report describes the effects of the V-ATPase inhibitor The vectorial transport of proteins from the endoplasmic concanamycin B, a close structural relative of the bafilomyreticulum to the cell surface for which no specific regulatory cins (Bowman et al., 1988),on the constitutive secretory as a compound sorting signals have beenidentified is often referred to as the pathway in HepG2 cells. Originally discovered with antiproliferative effects (Kinashi et al., 1984), it has constitutive secretory pathway (Palade, 1975). It is widely since been realized that i n vitro, concanamycin B, like bafiheld that such proteins move by default to theplasma memlomycin, is an extremely potent inhibitor (IC60 = 1-5 nM) of brane, unless specifically sorted to other compartments (Pfef- the H+-ATPases, with an almost perfect ability to discrimifer andRothman, 1987; Mellman and Simons, 1992). Cellular nate between the mitochondrial, plasma membrane, and vacsorting and targeting processes appear to rely on the internal uolar ATPases (Bowman et al., 1988; Mattsson et al., 1991; ionic environment of components of the endocytic and exo- Woo et al., 1992). We showthat exposure of HepG2 cells to cytic pathways (Mellman et aL, 1986;Anderson and Orci, low concentrations of concanamycin B significantly delays 1988; Mellman and Simons, 1992). The plasma membrane, the onset of secretion of the major proteins released by this mitochondria, and the vacuolar membrane system each pos- cell, without affecting protein synthesis. In intact cells, the sess unique electrogenic or non-electrogenicproton pumping inhibition is saturable at 50 nM of the drug. Movement of ATPases which lower intralumenal pH (Bowman et al., 1988; proteins from the endoplasmic reticulum to the Golgi complex Nelson, 1992a). Genetic experiments in the yeast Sacchuro- occurs at normal rates. Therefore, the delay appears to result myces cereuisiae have provided strong evidence implicating from inhibiting intra-Golgi trafficking or Golgi to plasma membrane delivery. The conversion of proalbumin to albumin * The costs of publication of this article were defrayed in part by is not affected by concanamycin B. In both intact and Strepthe payment of page charges. This article must therefore be hereby tolysin 0 (Strep 0)-permeabilized cells exposed to concanamarked ‘‘advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. These authors contributed equally to this study. Supported by Organon International bv, Oss, The Netherlands. 11 Present address: Central Research Laboratories of Ajinomoto Co. Inc. 1-1 Suzuki-chou, Kawasaki-ku, Kanagawa 210,Japan. ** To whom all correspondence should be addressed Center for Cancer Research, Dept. of Biology, Massachusetts Institute of TechFax: 617-253-9891. nology, Cambridge, MA 02139.Tel.: 617-253-0519;

The abbreviations used are: V-ATPase, vacuolar H+-ATPases; Strep 0, streptolysin 0; BFA, brefeldin A; GTPyS, guanosine 5’-30-(thi0)triphosphate; d-AT, d-antitrypsin; Endo H, endoglycosidase H PBS, phosphate-buffered saline; Staph A, Staphylococclls aureus; PAGE, polyacrylamide gel electrophoresis; 1-D, one-dimensional, IEF, isoelectric focusing; PIPES, 1,4-~iperazinediethanes~fonic acid.

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Inhibitory Effectsof Concanamycin B mycin B, N-linked glycan modifkations are incomplete, suggesting that Golgi-processing enzymes require vacuolar H+ATPase-dependent acidificationfor normal activity. Indirect immunofluorescence studies showthat after long exposureto concanamycin B (>90min), alterations in Golgi morphology occur, but these changes appear distinct from those induced by monensin or brefeldin A(BFA). EXPERIMENTAL PROCEDURES

Materials-Concanamycin B was obtained from Ajinomoto Co. (Kanagawa, Japan). A 10 p~ stock solution of concanamycin B was prepared in ethanol. [=S]Methionine (specific radioactivity >lo00 Ci/mmol) was obtained from Du Pont-New England Nuclear; fetal calf serum, Dulbecco's modified Eagle's medium, RPMI, penicillin/ streptomycin (100 X), and methionine-free media were from Life Technology, Inc.; rabbit antisera to human albumin, al-antitrypsin (al-AT) and transferrin were purchased from Cappel (Durham, NC). Endoglycosidase H (Endo H) andcreatine phosphate were purchased from Boehringer Mannheim. Reduced Strep 0 was purchased from Wellcome Diagnostics (Beckenham, United Kingdom). Creatine phosphate kinase and ATP were obtained from Sigma. Rabbit antildlCp was generously provided by Steve Podos and Dr. M. Krieger (Department of Biology, Massachusetts Institute of Technology, Cambridge, MA). Rabbit anti-a-mannosidase I1 was generously provided by Dr. K. Moremen (Department of Biochemistry, University of Georgia, Athens, GA). Cell Culture-The human hepatoma cell line HepG2 was grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mM glutamine, and 1/1000 dilution units/ml penicillin and 100 pg/ ml streptomycin. Cells were harvested by trypsinization and plated on 35- or 60-mm tissue culture dishes 48-72 h before each experiment. Metabolic Labeling of HepC2-Confluent (95-100%) monolayers were incubated in methionine-free RPMI media for 30 min at 37 "C to deplete the endogenous methionine pool. Typically, monolayers were metabolically labeled for 10 min with 25 pCi/ml of [%]methionine/dish, in 1ml of methionine-free media at 37 "C. Concanamycin B was added either at the time of the pulse or 30 min prior to the pulse. At the endof the pulse, a chase was performed with 1mM cold methionine, in the continued presence of concanamycin B. At indicated timesdishes were placed on ice, followingwhich the cell medium was removed and assayed for secreted protein. The monolayers were rinsed with ice-cold PBS and thenlysed in cold Nonidet P-40 lysis buffer (0.5% NonidetP-40, 50 mM Tris-HC1, pH 7.3, and 5 mM MgCl.4. Immunoprecipitation-Cell lysates were precleared with normal rabbit serum, and immune complexes were removed by adsorption to Staphylococcus aureus (Staph A). Sequential immunoprecipitations were carried out for 3 h with the different antisera. In some experiments, immunoprecipitations were done in parallel overnight. Immune complexes were washed as described (Burke et al., 1984) and prepared for SDS-PAGE or 1-D isoelectric focusing (Neefjes et al., 1986). Gels were fluorographed and dried before exposure and quantitation on a Fujix BAS-2000 Bio-Image Analyzer. Endoglycosidase H Digestion-Immunoprecipitates were resuspended in 20 pl of Endo H digestion buffer (50 mM sodium citrate, pH 5.5, containing 0.2% (w/v) SDS) and heated for 5 min at 95 "C. The immunoprecipitates were digested with 2 milliunits of Endo H at 37 'C for 6-20 h with constant shaking. Mock digestions lacking Endo H were carried out in parallel. The reactions were terminated by solubilization in SDS sample buffer. Subcellular Fractionation Experiments-100-mm dishes were labeled for 10 min with 50 pCi of [%]methionine in the presence or absence of 10 nM concanamycin B and chased with 1mM methionine for the times indicated. Cells were washed twice with cold PBS and once with homogenization buffer (250 mM sucrose in 10 mM TrisHC1, pH 7.4) (Balch et al., 1984). A 20% cell suspension was prepared and the cells disrupted by Dounce homogenization (-30 strokes), using a tight fitting pestle. Nuclei were removed by centrifugation at 1,OOO x g for 10min and the resultingpostnuclear supernatant adjusted to 1.3 M sucrose. 4.5 ml of the postnuclear supernatant was overlaid with a discontinuous sucrose gradient of 1.1 M sucrose (5 ml), and 0.6 M sucrose (3 ml), in 10mM Tris-HC1, pH 7.4. Gradients were spun for 2 h at 40,000 revolutions/min in a Beckman sW41 ultracentrifuge. 16 equal fractions were collected, and each fraction was lysed with 0.5% Nonidet P-40for 30 min on ice and then subjected to immunoprecipitation as described. The protein content of each

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fraction was determined by Bradford analysis (Bradford, 1976). Streptolysin 0 Permeabilization-Confluent (90%) HepG2 cells grown on 60-mm dishes were labeled for 10 min with 25 pCi of [%] methionine in the presence or absence of 10 nM concanamycin B. Cells were transferred to 0 "C and incubated with 0.5 unit/ml Strep 0 in permeabilization buffer (PB) (137 mM NaCl, 2.7 mM KCl, 2 mM EGTA, 1mM CaClz, 2 mM MgC12, 20mM PIPES, pH7.3) for 10 min, in the continued presence of concanamycin B. Unbound Strep 0 was removed by washing the cells twice with PB at 0 "C. Cells were incubated in transport buffer (115 mM KCl, 2.5 mM MgClz, 1 mM CaC12,2 mM EGTA, 25 mM HEPES, pH 7.3) supplemented with an ATP-generating system (2.2 mM ATP, 0.44 mM CTP, 22.2 mM creatine phosphate, 31.8 units/ml creatine phosphate kinase) and transferred to 37 "C to simultaneously initiate pore formation and transport processes (Tan et al., 1992). After a chase of 0-120 min, in the presence or absence of concanamycin B, the cells were transferred to 0 "C. The medium was collected and thecells were lysed in Nonidet P-40 lysis buffer. Albumin and al-AT were immunoprecipitated as described above. Immunofluorescence-HepG2 cells grown to -60% confluence on ethanol washed 12-mm square glass coverslips, were washed three times with PBS, and thenfixed with 3.7% formaldehyde in PBS for 30 min. Cells were incubated with 50 mM NH,C1 for 10 min to quench excess formaldehyde and then washed with PBS before permeabilization with 0.1% Triton X-100,0.02% SDS in PBS for 10 min. The glass coverslips were placed face down in 25 pl of blocking solution (5% fetal calf serum, 2.5% normal goat serum, 0.1% Triton X-100, 0.02% SDS, 0.02% NaNB,in PBS) for 15-30 min. Primary antibody (25 pl of 3 ng/pl rabbit anti-ldlCp or rabbit anti-a-mannosidaseI1 in blocking solution) was added for 90-120 min. Subsequently, secondary antibody, (20pl of 12 pg/ml fluorescein-conjugated goat I g G fraction to rabbit I g G in blocking solution) was added for 45-60 min. Between each incubation step, the coverslips were briefly rinsed three times in 100 ml of0.1% Triton X-100, 0.02% SDS in PBS. At the conclusion of the experiment, the coverslips were briefly rinsed three times with 100 ml of0.1% Triton X-100, 0.02% SDS in PBS and once with 100 ml of water, and mounted face down in 8 pl of Vinol gel with DABCO antiquench, on microscope slides. RESULTS

Concanumycin B Delays the Onset and Rate of Secretion in HepG2 Cells-We investigated the effects of the V-ATPase inhibitor concanamycin B on transport along the secretory pathway. HepG2 cells were exposed to increasing concentrations ofconcanamycinBduringa10-minpulseandthen chased in the continued presence of the inhibitor for 1 h. Secreted albuminand a1-AT were isolated by immunoprecipitation of labeled product and analyzed on SDS-PAGE (Fig. a).Inhibitionofsecretionisreadilyapparentat 10 nM concanamycin B (Fig. lA).A corresponding increase in cellassociated labeled albumin and a1-AT is detected (data not shown). Quantitation of the total labeled product secreted shows a>70% reduction in protein released over the range of concentrations tested (Fig. 1B). Inhibition appears saturable at 50 nM concanamycin B. No effect on protein synthesis is detected in cells treated with the inhibitor (data notshown). Additionally,repeatedwashingofconcanamycinB-treated cells after the pulse does not reverse the inhibition (data not shown). These data show that inhibiting V-ATPases affects thetimelyreleaseofproteinswhichtraversetheexocytic pathway in HepG2 cells. We monitored the effects of concanamycin B on the biosynthesis of secretory proteins by pulse-chase experiments. Confluent cells were pulse labeled for 10 min either in the presence or absence of 10 nM concanamycin B, and a chase wasperformedforthe timesindicated(Fig. 2). Releaseof individual serum proteins occurs at characteristic rates due to differences in the rate of endoplasmic reticulum to Golgi al., 1983; Fries et al., 1984). Thusalbumin transport (Lodish et and al-AT are released at a much faster rate (Fig. 2, lane 5) than transferrin (Fig. 2, lane 7). When cells are exposed to concanamycinB, a marked delay in both the onset of secretion

Inhibitory Effects of Concanamycin B

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which they exitat a slower rate, an observation that suggests the probability of missorting upon dissipation of pH gradients in thesecretory pathway. Exposure to theV-ATPase inhibitor induces significant alterations in the ratesof secretion of the Concanamycin B (nM) 0 10 50 100 major soluble proteins in HepGZ cells. ER to Golgi Transport Occurs at Characteristic Rates in the Presence of Concanamycin B-The possibility that surface Albumin deposition was delayed due to impaired endoplasmic reticulum to Golgi transport was investigated. Although the pH of the endoplasmic reticulum is not known, it is suggested to be close to neutral, and to date no V-ATPaseshave been localized al-AT 0 to this region of the vacuolar system (Mellman et al., 1986). We initially scored for transport from the endoplasmic reticulum to the medial-Golgi by analyzing loss of sensitivity to Endo H (Kornfeld and Kornfeld, 1985). Immunoprecipitates of the cell-associated pools of al-AT and transferrinfrom a typical pulse-chase experiment, as described in Fig. 2, were subjected to Endo H digestion and subsequently analyzed on SDS-PAGE. Conversion of the immature, Endo H-sensitive B precursors to Endo H-resistant, complex product occurs with nearly identical rates in concanamycin B-treated cells and control cells (Fig. 4). Preincubating cells with concanamycin B for up to 90 min does not change the rate of conversion of Albumin Endo H-sensitiveprecursors to Endo H-resistant product, a1-AT indicating that thetime of addition of the drug does not affect endoplasmic reticulum to Golgi transfer (data not shown). Depletion of a1-AT seen in the lysates of control cells is due to secretion (Fig. 4, lanes 7 and 9). Only the complex-modified forms of al-AT and transferrin are released (data notshown) (Lodish et dl., 1983). The kinetics of loss of Endo H sensitivity m ” 0 were analyzed quantitatively and found to be similar for Iconcanamycin B-treated cells and control cells (datanot 0 shown). Short chase times indicatea 5-min delay in the acquisition of Endo H resistance for al-AT in the presence C 0 of concanamycin B (Fig. 4, lanes 3 and 4).* This is most likely due to concanamycin B-induced inhibition of Golgi processing enzyme activities, rather thandelayed endoplasmic reticulum to medial-Golgi transport. Faster migrating mature forms of a1-AT and of transferrin are produced in the presence of concanamycin B. Most significantly, it is this form of al-AT that persists in thecells exposed to theinhibitor. An increase in concanamycin B concentration from 10 to50 nM does not induce any further shifts in mobility of al-AT (Fig. 1, data 0 10 50 100 not shown). Subcellular fractionation experiments designed to enrich Concanamycin B (nM) FIG. 1. A, concanamycin B inhibits secretion in HepG2 cells. for Golgi fractions (see Balch et al., 1984) supported the Confluent monolayers were pulse labeled with 25 pCi of [35S]methi- interpretation thatendoplasmic reticulum to Golgi transport onine for 10 min in the presence of the indicated concentrations of was not affected by treatment with concanamycin B. After concanamycin B. A chase was performed with 1 mM cold methionine zero min of chase, over 85% of newly synthesized a1-AT in for 60 min in the continued presence of the inhibitor and the exper- both control cells and concanamycin B-treated cells is found iments processed as described under“Experimental Procedures.” associated with the dense fractions, and hence resides in the Albumin and a1-AT in the medium were isolated by immunoprecipitation and analyzed on SDS-PAGE. B, quantitation of secreted endoplasmic reticulum (Fig. 5 A ) . Only immature high manproduct. Labeled products in the medium and cell lysate were quan- nose presursors, which are sensitive to Endo H, are evident titated as described under “Experimental Procedures.” The fraction (data notshown; see also Fig. 4). Following a chaseof 30 min, of total albumin or al-AT secreted a t each concentration of concan- asimilarcharacteristic shiftin sedimentation, suggesting amycin B is shown. endoplasmic reticulum to Golgi transfer has occurred, is detected in both control cells and cells exposed to the inhibitor and in the quantitative release of all three products is observed (Fig. 5B). (Similardistributions were detected for serum (Fig. 2, lanes 5-8). Analysis of the pulse-chase experiment albumin (data not shown).) About 60% of the total d - A T from Fig. 2, shown in Fig. 3, suggests that concanamycin B sediments with Golgi membranes in control and concanamyinduces a delay inthe onset of secretion,with maximal cin B-treated cells (Fig. 5B). Thisproduct has acquired comdecrease observed at 60 min of chase for albumin and a1-AT plex modifications andisresistanttoEndo H (data not (Fig. 3,A and B) and 120 at min of chase fortransferrin. The shown). The dense fractions contain predominantly Endo Hfraction of total protein secreted in thepresence of concana- sensitive al-AT (data not shown). Since the conversion of mycin B does not reach control levels, even after the longest high mannose precursors to complex type glycoproteins, as chase time tested (Fig. 3). It appears that a fraction of the soluble proteins is delivered to intracellular stations from * M. Yilla, A. Tan, andH. L. Ploegh, unpublished data.

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FIG.2. Concanamycin B delays the onset of secretion of albumin, al-AT, and transferrin. HepG2 cells were pulse labeled for 10 min with 25 pCi of [%]methionine, in thepresence (-) or absence (+) of 10 nM concanamycin B. A chase was performed for indicated times in thecontinued presence or absence of the inhibitor. Albumin, al-AT, andtransferrin in the medium were isolated by immunoprecipitation and analyzed on SDS-PAGE.

well as localization of product by subcellular fractionation experiments appear similar under normal and inhibitory conditions, we conclude that secretory proteins in the presence of concanamycin B reach the Golgi at a rate indistinguishable from that seen in control cells. Concanamycin B-induced Inhibition of N-Linked Glycan Modifications in Intact and Permeabilized HepG2CellsFaster migrating forms of both secreted (Fig. 2), and intracellular (Fig. 6 A , Intact cells), al-AT and transferrin, indicative of incomplete N-linked glycan modifications,are produced in concanamycin B-treated intact cells. This suggests that inhibiting acidification by V-ATPases affects the activity of Golgi-processing enzymes. Westudied the effects of concanamycin B on glycosylation patterns of a1-AT in Strep 0permeabilized cells. In the permeabilized cell system, transport and glycosylation processes remain functional, but the al-AT produced is not completely mature (Tan et al., 1992), and therefore migrates faster than a1-AT synthesized in intact cells (Fig. 6). In Strep 0-permeabilized cells, 0.1 nM concanamycin B is sufficient to induce a delay in the onset of secretion of the soluble proteins (data not shown). In such to the extracellular permeabilizedcells,compoundsadded medium gain access to their target structures without delay. When Strep 0-permeabilized cells are exposed to 10 nM concanamycin B, a form of a1-AT is produced which has an electrophoretic mobility identical to the high mannose precursor in its most extensively trimmed form (Fig. 6A, Permeabilized Cells). This product is never detected in intact cells, or their secretions, even at thehighest concentration of concanamycin B tested (Fig. 1,data not shown). This form of a1-AT is resistant to Endo H treatment and hence must reside in the medial-Golgi or beyond (Fig. 6B, Permeabilized Cells). The appearance of this aberrantly processed form of a1-AT suggests that the effects of concanamycin B on Nlinked glycan modificationsin semi-intact cells, and in intact cells, are distinct. This difference is likely due to facilitated access of concanamycin B to vacuolar ATPases, in the absence of the plasma membrane barrier. To examine the extent of sialylation, al-AT produced in concanamycin B-treated intact and permeabilized cells was analyzed by 1-D IEF. Exposure of intact cells to concanamycin B results in incomplete addition of terminal sialic acids to a1-AT (Fig. 7A, Intact Cells). The species which persist

contains sialic acids, but the patternof distribution is altered (quantitation not shown). Whenpermeabilizedcells are treated with the inhibitor, a total reduction in the number of sialic acids transferred to a1-AT occurs (Fig. 7A, Permeabilized Cells). Quantitation of the distribution of sialic acids reveals that the shift in sialylation patterns induced by concanamycin B is not identical for intact or permeabilized cells (data not shown). It appears that the delivery of concanamycin B to its target(s)promoted by permeabilization of HepG2 cells results in a more pronouncedimpairment of Golgi processing enzyme activity. We examined the effects of concanamycin B on proteolytic conversion of proalbumin to albumin in both the intact cell and the permeabilized cellby 1-D IEF analysis. No effect on this conversion was detected and the ratio of proalbumin to albumin remained similar under all conditions (Fig. 7B, quantitation not shown). This suggests that the delay in albumin secretion induced by the V-ATPase inhibitor is not due to delayed maturation events. Because GTPyS ismembrane impermeant, its effects ona1-AT maturation and proalbumin conversion necessitate the use of a semi-intact cell systemas done here by permeabilization with Strep 0. The addition of GTPyS (shown to blockendoplasmicreticulum to Golgi transport, Tan et al., 1992) to Strep 0-permeabilized cells significantly impaired the conversion of proalbumin to albumin (Fig. 7 B ) . Therefore, transfer of proalbumin from the endoplasmic reticulum to a subsequent compartment is required for its proteolytic conversion. Concanamycin B Induces Slight Alterations in Golgi Morphology-Indirect immunofluorescence techniques were used to examine Golgimorphology in concanamycin B-treated cells. Two Golgi markers, a-mannosidase I1 (a lumenal Golgi protein) (Moremen and Touster, 1986) and ldlCp (peripherally associated with Golgi membra ne^)^ were used to probe Golgi structures. The immunofluorescence staining patterns for these two antibodies colocalize with Golgi structures in control cells and couldbeused interchangeably (data not shown). The effects of concanamycin B were compared with those of the carboxylic ionophore monensin. After 15min of exposure, the morphology of the Golgi in concanamycin Btreated cells appears similar to that seen in control cells (Fig. S. R. Podos, J. Reddy, J. Ashkenas, and M. Krieger, manuscript in preparation.

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Fractlon # Frectlon # FIG.5. Concanamycin B does not alter the intracellular distributionof aVl-AT in HepG2 cells. Cells were pulse labeled for 10 min in the presence or absence of inhibitor and chased as indicated. A postnuclear membrane fraction was prepared and centrifuged through a discontinuous sucrose gradient. The gradient fractionscollected were subsequently immunoprecipitated with antiserum to a1-AT. Analysis of the totalprotein distributionacross the gradient reveals two distinct peaks, a large peak between fractions 4-7 and a smaller peak between fractions 10-14 (data not shown). Labeled proteins at the1.3/1.1 interface (fraction 6 )were contained in the dense endoplasmic reticulum or PM vesicles and those at the 1.1/0.6 interface (fraction 12) were contained in lighter Golgi membranes (Balch et al., 1984). The data are averaged over two separate experiments. The incorporated radioactivity from individual fractions is combined into endoplasmic reticulum pools (fractions1-6) or Golgi pools (fractions 7-14) as referred to in the text.

8, A and B ) (The staining pattern was similar for concanamycin B-treated cells probed with l d c p (data not shown.) Characteristic staining is evident. H ~ in cells treated with monensin, vacuolizationof the Golgi is

posed to BFA are shown for comparison (Fig. 8, E and D, respectively). BFA is includedas an agent which inducesa 8coat dissociation ~ protein-like ~ ~ ~ (orci~et al., 1991; , Donaldson et az.9 199119 of l a c p from Golgi-membranes.3Although the effects of concanamycin B are distinct from those of monensin and BFA, Golgi structures are nonetheless slightly altered.

apparent after l5 min Of exposure (Fig. 8E),consistent with the known effects of monensin on Golgi morphology (Tartakoff, 1983). At later times, we do detect some morphoDISCUSSION logical disruption of Golgi structures in cells exposedto conWe have studied the effects of concanamycinB, an inhibitor canamycin B (Fig. 8C).Monensin-treated cells and cells ex- of H+-ATPases of the vacuolar system, on the secretory

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FIG. 6. N-Linked glycan modifications are incomplete in intact and permeabilizedHepG2 cells exposed to concanamycin B. Intact (Intact Cells) or Strep-0-permeabilized cells (Permobilized Cells) were pulse labeled for 10 min with 25 pCi of [%+nethionine in the presence or absence of concanamycin B and chased for times indicated in the continued presence or absence of the inhibitor. As a control for permeabilization efficiency, cells were chased for 60 min in the presence of1.0 mM GTP-yS which blocks endoplasmic reticulum exit in permeabilized cells. al-AT isolated by immunoprecipitation from the cell lysates was analyzed either directly ( A ) ,or first digested with Endo H ( B ) ,and subsequently analyzed on 10% SDS-PAGE. The migration positions of al-AT with high mannose type (HM)or complex type ( C ) oligosaccharides, and Endo H-resistant (Endo HR)and Endo H-sensitive (Endo P)are indicated.

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FIG. 7. A, concanamycin B inhibits sialylation of al-AT. Immunoprecipitates of cell associated al-AT from the experiments described in Fig. 6 were analyzed by one dimensional-IEF. B, conversion of proalbumin to albumin is unaffected by concanamycin B. 1-D IEF analysis of albumin immunoprecipitates from the experiments described in Fig. 6 are shown in B. The sialylated forms of al-AT and the position of proalbumin and albumin are indicated.

pathway in HepG2 cells. This report shows that treating cells with concanamycin B triggers a delay in the onset and rate of secretion and inhibits N-linked glycan modifications of soluble proteins. Since treatment with concanamycin B prevents intralumenal acidification by V-ATPases (Woo et al., 1992),we propose that acidification by V-ATPases is required to control timely exit along the secretory pathway and to complete carbohydrate modifications. Because endoplasmic reticulum to Golgi transport in the presence of concanamycin B occurs at rates similar to those seen in control cells, we suggest that the block in the exocytic pathway occurs at the tram-Golgiltram-Golgi network (TGN). The TGN is mildly

acidic, pH 2 6 and is generally regarded to be the sorting station for delivery of proteins to cellular compartments (Orci et aZ., 1984; Griffiths and Simons, 1986; Mellman and Simons, 1992). Inadequate acidification of the TGN may result in delayed protein sorting and packaging. As a consequence, secretory proteins are released into the surrounding medium at a reduced rate. Alternatively, blocking acidification could cause a default pathway to be utilized. In this case, proteins would be shunted into a different pathway which still results in surface deposition and/or secretion as in normal cells, but at a lower rate. A “missorting” model impliesthat more than one pathway exists for TGN to PM trafficking for soluble

Inhibitory Effects of Concanamycin B

19099

A

control

B

15 min concanamycin B

C

90 min concanamycin B

D

90 min brefeldin A

E

15 min monensin

F

90 min monensin

FIG. 8. Morphology of the Golgi apparatusis slightly altered after exposure to concanamycinB. HepG2 cells treated asindicated were prepared for immunofluorescence as described under “Experimental Procedures.” Golgi morphology was analyzed with either anti-amannosidase I1 ( B ) or with anti-ldlCp (A, C-F). Control cells ( A ) ;concanamycin B-treated cells, 15 min ( B ) or 90 min ( C ) ;monensin (25 pM)-treated cells, 15 min ( E )or 90 min (F); brefeldin A-treated cells, 90 min (D).

proteins. Studies in yeast have shown that mutations in VATPases can result in protein missorting along the vacuolar system (Yamashiro et al., 1990; Klionsky et al., 1992). These reports haveprovided the strongest evidence so far that vacuolar ATPases play a role in efficient and correct sorting of proteins to theyeast vacuole. Deliveryto thevacuole, which resembles the mammalian lysosome, is altered by protonophores, acidotropic agents, and by bafilomycin (Banta et al., 1988; Klionsky et al., 1990).It was proposedthat interference with the activity of V-ATPases results in missorting in the Golgi complex or in post-Golgi vesicles(Nelson, 1992b). Our data, obtained in mammalian cells, would not be inconsistent with such proposals. It has been inferred from previous work that sialyltransferases located in the trans-Golgi and TGN(Duncan and Kornfeld, 1988) mayrequire an acidic pH for activity (Pohlentz et al., 1988). Inhibition of N-linked glycan modifications have also been reported in studies of mammalian cells which have defects in vacuolar acidification (Barasch et al., 1991; Laurie and Robbins, 1991), and in cells exposed to chloroquine and ammonium chloride (Thorens andVassalli, 1986).This report shows that specific inhibition of V-ATPases by concanamycin

B results in suppression of N-linked glycan modifications in both intact and permeabilized cells. Therefore, acidification of Golgi compartments is necessary to complete carbohydrate modifications. Analysis of intact cells exposed to concanamycin B suggests that sialic acids are transferred to al-AT but the extent of sialylation is reduced when compared to control. Implicit in this statement is the assumption that the substrate ( i e . CMP-NeuAc) is not limiting under the given conditions in intact cells, an assumption that was not verified experimentally. In the semi-intact system, inhibition of complex-type glycan modifications, and of sialic acid transfer in particular, is far more pronounced indicating that permeabilization promotes access of concanamycin B to its target(s). Under these conditions, the majority of a1-AT is non-sialylated. A V-ATPase was isolated from rat liver Golgi and shown to be inhibited by the macrolide antibiotic bafilomycin in vitro (Moriyama and Nelson, 1989). It is possible that more than one type of V-ATPase may reside in the Golgi with all of them not equally susceptible to inhibition by concanamycin B. In the permeabilized cell, local pH may be altered even more drastically as putative additional V-ATPases are inhibited. The reduction in complex-type N-linked glycan modifi-

19100

Inhibitory Effects of Concanamycin B

cations, particularly sialylation, may stem from an effect of post-translational modifications. pH on activity of the transferases themselves, or on the Acknowledgments-We thank Drs. Monty Krieger, Kelly Moreavailability of the nucleoside-sugar substrates. It is conceivable that the ApH in the tram-Golgi contributes to delivery men, and Steve Podos for generously providing reagents and Drs. of substrate or efflux of nucleoside phosphates (Hirschberg Jeremey Auey, Monty Krieger, Harvey Lodish, Ton Schumacher, and Steve Podos for critically evaluating this manuscript and for and Snider, 1987). Thus a dissipation ofApH could prove their helpful discussions. inhibitory in a less direct manner to reactions catalyzed by REFERENCES glycosyltransferases. R. G. W., and Orci, L. (1988) J. Cell B i d 106,539-543 Most of the organelles of the vacuolar system contain H+- Anderson R. G. W., and Pathak,R. K. 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’P. Benaroch, M. Yilla, and H. L. Ploegh, unpublished data.

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