THE JOURNAL 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+-ATPases in 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 &Center for Cancer Research. Demrtment of Bwbgy, Massachusetts Institute of Techmbgy, , . Cambridgi Massachusetts 02139

The macrolide antibiotic concanamycin B is a highly selective inhibitor (IC6,, = 5 nM) of the H+-ATPases of the vacuolar system. We have examined the effects of concanamycin B on the constitutive secretory pathway of the human hepatoma cell line, HepG2. In cells ex- p d to 10 n M concanamycin B, transport from the endoplasmic reticulum to the Golgi occurs at normal ratee, as determined by pulse-chase analysis of endo- glycosidase H-sensitive product in conjunction with subcellular fractionation experiments. However, in- tra-Golgi trafficking or Golgi to plasma membrane delivery is significantly impaired. A delay in the onset of secretion of the major secretory proteins, albumin, al-antitrypsin and transferrin is observed. Processing of N-linked glycans by sialyltransferases is inhibited, resulting in secreted glycoproteins which are modified less extensively. In view of the acidic pH of the trum- Golgi and the trum-Golgi network, these studies sug- gest that acidification by vacuolar ATPases is critical to achieving timely secretion and correct N-linked gly- can modifications of proteins which follow the consti- tutive secretory pathway.

The vectorial transport of proteins from the endoplasmic reticulum to the cell surface for which no specific regulatory sorting signals have been identified is often referred to as the constitutive secretory pathway (Palade, 1975). It is widely held that such proteins move by default to the plasma mem- brane, unless specifically sorted to other compartments (Pfef- fer and Rothman, 1987; Mellman and Simons, 1992). Cellular sorting and targeting processes appear to rely on the internal ionic environment of components of the endocytic and exo- cytic pathways (Mellman et aL, 1986; Anderson and Orci, 1988; Mellman and Simons, 1992). The plasma membrane, mitochondria, and the vacuolar membrane system each pos- sess unique electrogenic or non-electrogenic proton pumping ATPases which lower intralumenal pH (Bowman et al., 1988; Nelson, 1992a). Genetic experiments in the yeast Sacchuro- myces cereuisiae have provided strong evidence implicating

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘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 Tech- nology, Cambridge, MA 02139. Tel.: 617-253-0519; Fax: 617-253-9891.

the vacuolar H+-ATPases (V-ATPases)’ in particular, in sort- ing of soluble proteins to the vacuole, the counterpart of the mammalian lysosome, and in sorting along the vacuolar sys- tem (Yamashiro et al., 1990; Klionsky et al., 1992). Several studies have suggested a role for acidified compartments in membrane trafficking in mammalian cells (reviewed in Mell- man et al., 1986; Anderson and Orci, 1988; Laurie and h b - bins, 1991). Acidotropic agents such as chloroquine, prima- quine and ammonium chloride, and ionophores such as mo- nensin have been commonly used to study pH regulation (Uchida et al., 1980; Strous et al., 1985; Mellman et al., 1986). Although these agents have found widespread use in manip- ulating the pH of intracellular acidic organelles, the high concentrations required to achieve desired effects, along with the secondary nonspecific manifestations of these compounds, have limited the scope of their usefulness (Mellman et al., 1986). Similarly, ionophores such as monensin can dissipate ionic gradients across membranes, but their selectivity is not absolute and gross morphological alterations, particularly of the Golgi apparatus, occur (Tartakoff, 1983; Mellman et al., 1986). Macrolide antibiotics that selectively block the func- tion of distinct types of H+-ATPases have created new pos- sibilities for exploring the role of the acidic pH of the trans- Golgi region.

This report describes the effects of the V-ATPase inhibitor concanamycin B, a close structural relative of the bafilomy- cins (Bowman et al., 1988), on the constitutive secretory pathway in HepG2 cells. Originally discovered as a compound with antiproliferative effects (Kinashi et al., 1984), it has since been realized that in vitro, concanamycin B, like bafi- lomycin, is an extremely potent inhibitor (IC60 = 1-5 nM) of the H+-ATPases, with an almost perfect ability to discrimi- nate between the mitochondrial, plasma membrane, and vac- uolar ATPases (Bowman et al., 1988; Mattsson et al., 1991; Woo et al., 1992). We show that exposure of HepG2 cells to low concentrations of concanamycin B significantly delays the onset of secretion of the major proteins released by this cell, without affecting protein synthesis. In intact cells, the inhibition is saturable at 50 nM of the drug. Movement of proteins from the endoplasmic reticulum to the Golgi complex occurs at normal rates. Therefore, the delay appears to result from inhibiting intra-Golgi trafficking or Golgi to plasma membrane delivery. The conversion of proalbumin to albumin is not affected by concanamycin B. In both intact and Strep- tolysin 0 (Strep 0)-permeabilized cells exposed to concana-

The abbreviations used are: V-ATPase, vacuolar H+-ATPases; Strep 0, streptolysin 0; BFA, brefeldin A; GTPyS, guanosine 5’-3- 0-(thi0)triphosphate; d-AT, d-antitrypsin; Endo H, endoglycosi- dase H PBS, phosphate-buffered saline; Staph A, Staphylococclls aureus; PAGE, polyacrylamide gel electrophoresis; 1-D, one-dimen- sional, IEF, isoelectric focusing; PIPES, 1,4-~iperazinediethanes~- fonic acid.

19092

Inhibitory Effects of Concanamycin B 19093

mycin B, N-linked glycan modifkations are incomplete, sug- gesting that Golgi-processing enzymes require vacuolar H+- ATPase-dependent acidification for normal activity. Indirect immunofluorescence studies show that after long exposure to concanamycin B (>90 min), 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) and creatine 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 anti- ldlCp 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 pro- vided 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 [%]methi- onine/dish, in 1 ml of methionine-free media at 37 "C. Concanamycin B was added either a t the time of the pulse or 30 min prior to the pulse. At the end of the pulse, a chase was performed with 1 mM cold methionine, in the continued presence of concanamycin B. At indi- cated times dishes were placed on ice, following which the cell medium was removed and assayed for secreted protein. The monolayers were rinsed with ice-cold PBS and then lysed in cold Nonidet P-40 lysis buffer (0.5% Nonidet P-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 experi- ments, immunoprecipitations were done in parallel overnight. Im- mune 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 quan- titation on a Fujix BAS-2000 Bio-Image Analyzer.

Endoglycosidase H Digestion-Immunoprecipitates were resus- pended 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 la- beled for 10 min with 50 pCi of [%]methionine in the presence or absence of 10 nM concanamycin B and chased with 1 mM methionine for the times indicated. Cells were washed twice with cold PBS and once with homogenization buffer (250 mM sucrose in 10 mM Tris- HC1, 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 10 min and the resulting postnuclear 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 10 mM 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-40 for 30 min on ice and then subjected to immunoprecipitation as described. The protein content of each

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, 1 mM CaClz, 2 mM MgC12, 20 mM PIPES, pH 7.3) 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 the cells 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 then fixed 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 permeabili- zation 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-mannosidase I1 in blocking solution) was added for 90-120 min. Subsequently, second- ary antibody, (20 pl 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 of 0.1% Triton X-100, 0.02% SDS in PBS. At the conclusion of the experiment, the coverslips were briefly rinsed three times with 100 ml of 0.1% 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 concentra- tions of concanamycin B during a 10-min pulse and then chased in the continued presence of the inhibitor for 1 h. Secreted albumin and a1-AT were isolated by immunoprecip- itation of labeled product and analyzed on SDS-PAGE (Fig. a). Inhibition of secretion is readily apparent at 10 nM concanamycin B (Fig. lA). A corresponding increase in cell- associated 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 not shown). Additionally, repeated washing of concanamycin B-treated cells after the pulse does not reverse the inhibition (data not shown). These data show that inhibiting V-ATPases affects the timely release of proteins which traverse the exocytic pathway in HepG2 cells.

We monitored the effects of concanamycin B on the bio- synthesis 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 was performed for the times indicated (Fig. 2). Release of individual serum proteins occurs at characteristic rates due to differences in the rate of endoplasmic reticulum to Golgi transport (Lodish et al., 1983; Fries et al., 1984). Thus albumin 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 concanamycin B, a marked delay in both the onset of secretion

19094 Inhibitory Effects

A

Concanamycin B (nM) 0 10 50 100

Albumin J”

al-AT 0

B

- m 0 ” I- - 0

C 0 -

Albumin a1 -AT

0 1 0 5 0 1 0 0

Concanamycin B (nM) FIG. 1. A, concanamycin B inhibits secretion in HepG2 cells.

Confluent monolayers were pulse labeled with 25 pCi of [35S]methi- onine for 10 min in the presence of the indicated concentrations of concanamycin B. A chase was performed with 1 mM cold methionine for 60 min in the continued presence of the inhibitor and the exper- iments processed as described under “Experimental Procedures.” Albumin and a1-AT in the medium were isolated by immunoprecip- itation and analyzed on SDS-PAGE. B, quantitation of secreted product. Labeled products in the medium and cell lysate were quan- titated as described under “Experimental Procedures.” The fraction of total albumin or al-AT secreted at each concentration of concan- amycin B is shown.

and in the quantitative release of all three products is observed (Fig. 2, lanes 5-8). Analysis of the pulse-chase experiment from Fig. 2, shown in Fig. 3, suggests that concanamycin B induces a delay in the onset of secretion, with maximal decrease observed at 60 min of chase for albumin and a1-AT (Fig. 3, A and B) and at 120 min of chase for transferrin. The fraction of total protein secreted in the presence of concana- mycin B does not reach control levels, even after the longest chase time tested (Fig. 3). It appears that a fraction of the soluble proteins is delivered to intracellular stations from

of Concanamycin B

which they exit at a slower rate, an observation that suggests the probability of missorting upon dissipation of pH gradients in the secretory pathway. Exposure to the V-ATPase inhibitor induces significant alterations in the rates of secretion of the major soluble proteins in HepGZ cells.

ER to Golgi Transport Occurs at Characteristic Rates in the Presence of Concanamycin B-The possibility that surface 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-ATPases have been localized to this region of the vacuolar system (Mellman et al., 1986). We initially scored for transport from the endoplasmic retic- ulum 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 transferrin from 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 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 Endo H-sensitive precursors to Endo H-resistant product, indicating that the time 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 not shown) (Lodish et dl., 1983). The kinetics of loss of Endo H sensitivity were analyzed quantitatively and found to be similar for concanamycin B-treated cells and control cells (data not shown). Short chase times indicate a 5-min delay in the acquisition of Endo H resistance for al-AT in the presence 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 than delayed 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 the cells exposed to the inhibitor. An increase in concanamycin B concentration from 10 to 50 nM does not induce any further shifts in mobility of al-AT (Fig. 1, data not shown).

Subcellular fractionation experiments designed to enrich for Golgi fractions (see Balch et al., 1984) supported the interpretation that endoplasmic reticulum to Golgi transport was not affected by treatment with concanamycin B. After zero min of chase, over 85% of newly synthesized a1-AT in both control cells and concanamycin B-treated cells is found associated with the dense fractions, and hence resides in the endoplasmic reticulum (Fig. 5A) . Only immature high man- nose presursors, which are sensitive to Endo H, are evident (data not shown; see also Fig. 4). Following a chase of 30 min, a similar characteristic shift in sedimentation, suggesting endoplasmic reticulum to Golgi transfer has occurred, is de- tected in both control cells and cells exposed to the inhibitor (Fig. 5B). (Similar distributions were detected for serum albumin (data not shown).) About 60% of the total d - A T sediments with Golgi membranes in control and concanamy- cin B-treated cells (Fig. 5B). This product has acquired com- plex modifications and is resistant to Endo H (data not shown). The dense fractions contain predominantly Endo H- sensitive al-AT (data not shown). Since the conversion of high mannose precursors to complex type glycoproteins, as

* M. Yilla, A. Tan, and H. L. Ploegh, unpublished data. ~~

Inhibitory Effects of Concanumycin B 19095

Chase (min) 240 120 60 30 10 0

Concanamycin B + + + + + + -

Albumin 0"-

al-AT - Transferrin 0 - --- "

1 2 3 4 5 6 I 8 9 10 11 12

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 the presence (-) or absence (+) of 10 nM concanamycin B. A chase was performed for indicated times in the continued presence or absence of the inhibitor. Albumin, al-AT, and transferrin 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 con- ditions, 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 HepG2 Cells- Faster migrating forms of both secreted (Fig. 2), and intracel- lular (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 in- hibiting acidification by V-ATPases affects the activity of Golgi-processing enzymes. We studied the effects of concan- amycin B on glycosylation patterns of a1-AT in Strep 0- permeabilized cells. In the permeabilized cell system, trans- port 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 permeabilized cells, compounds added to the extracellular 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 pre- cursor in its most extensively trimmed form (Fig. 6A, Per- meabilized Cells). This product is never detected in intact cells, or their secretions, even at the highest 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 N- linked glycan modifications in 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 concanamy- cin 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 pattern of distribution is altered (quantitation not shown). When permeabilized cells are treated with the inhibitor, a total reduction in the number of sialic acids transferred to a1-AT occurs (Fig. 7A, Permeabil- ized Cells). Quantitation of the distribution of sialic acids reveals that the shift in sialylation patterns induced by con- canamycin B is not identical for intact or permeabilized cells (data not shown). It appears that the delivery of concanamy- cin B to its target(s) promoted by permeabilization of HepG2 cells results in a more pronounced impairment of Golgi proc- essing enzyme activity.

We examined the effects of concanamycin B on proteolytic conversion of proalbumin to albumin in both the intact cell and the permeabilized cell by 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, quan- titation 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 is membrane impermeant, its effects on a1-AT maturation and proalbumin conversion necessitate the use of a semi-intact cell system as done here by permeabilization with Strep 0. The addition of GTPyS (shown to block endoplasmic reticulum to Golgi transport, Tan et al., 1992) to Strep 0-permeabilized cells significantly impaired the conversion of proalbumin to albu- min (Fig. 7 B ) . Therefore, transfer of proalbumin from the endoplasmic reticulum to a subsequent compartment is re- quired for its proteolytic conversion.

Concanamycin B Induces Slight Alterations in Golgi Mor- phology-Indirect immunofluorescence techniques were used to examine Golgi morphology in concanamycin B-treated cells. Two Golgi markers, a-mannosidase I1 (a lumenal Golgi protein) (Moremen and Touster, 1986) and ldlCp (peripher- ally 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 could be used interchangeably (data not shown). The effects of concanamycin B were compared with those of the carboxylic ionophore monensin. After 15 min of exposure, the morphology of the Golgi in concanamycin B- treated cells appears similar to that seen in control cells (Fig.

S. R. Podos, J. Reddy, J. Ashkenas, and M. Krieger, manuscript in preparation.

19096

A

loo

eo

40

20

01

Inhibitory Effects of Concanamycin B

I ”- ” c m m * m 0

100 200

Chase (mln)

C

W

W

20

0

’~ ( 0 80 240 /

100 200

Chase (mln)

Chase (mln) FIG. 3. The rate of secretion of albumin, (rl-AT, and transferrin is altered in the presence of concanamycin B. Labeled

products in the medium and cell lysate from the experiment shown in Fig. 2 were quantitated as described under “Experimental Procedures.” The percent total product secreted at each chase point is shown. The graphs (insets) represent the decrease in labeled product secreted at each chase point, as a result of concanamycin B treatment. Maximal decrease is observed at 60 min of chase for albumin ( A ) and d - A T ( B ) , and at 120 min of chase for transferrin (C).

Inhibitory Effects of Concanamycin B

Chase (min) 240 120- 60 30 10 0

Coneanamyein B + + + + + + -

19097

al-AT

Transferrin

-Endo HR

”- -, -Endo Hs

- ”- -Endo HR

m - -EndoHS

1 2 3 4 S 6 1 8 9 10 11 12

FIG. 4. Endoplasmic reticulum to medial-Golgi transport is not altered by concanamycin B. al-AT and transferrin immunopre- cipitates from a typical pulse-chase experiment as described in the legend of Fig. 2 were subjected to digestion with Endo H as described under “Experimental Procedures.” Endo Hs, precursor products sensitive to Endo H Endo HR, complex products resistant to Endo H.

A B

1 0 rnln pulse -[3- Cm.d 0 min ehoso -W- -&e

20

- Q 10

0 c c

0

10 rnln pulw -Q- M d 50 mln ohmso “c. ~ . “ - , & e

0 1 2 3 4 5 8 7 8 0 1 0 1 1 1 2 1 3 1 4 1 5 1 6

Frectlon # Fractlon #

Vl-AT in HepG2 cells. Cells were pulse labeled for FIG. 5. Concanamycin B does not alter the intracellular distribution of a 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 fractions collected were subsequently immunoprecipitated with antiserum to a1-AT. Analysis of the total protein distribution across 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 the 1.3/1.1 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 (fractions 1-6) or Golgi pools (fractions 7-14) as referred to in the text.

8, A and B ) (The staining pattern was similar for concana- posed to BFA are shown for comparison (Fig. 8, E and D, mycin B-treated cells probed with l d c p (data not shown.) respectively). BFA is included as an agent which induces a 8- Characteristic staining is evident. H ~ ~ ~ ~ ~ ~ , in coat protein-like dissociation (orci et al., 1991; Donaldson et

effects of concanamycin B are distinct from those of monensin cells treated with monensin, vacuolization of the Golgi is az.9 199119 of l a c p from Golgi-membranes.3 Although the

apparent after l5 min Of exposure (Fig. 8E), consist- and BFA, Golgi structures are nonetheless slightly altered. ent with the known effects of monensin on Golgi morphology (Tartakoff, 1983). At later times, we do detect some morpho- DISCUSSION logical disruption of Golgi structures in cells exposed to con- We have studied the effects of concanamycin B, an inhibitor canamycin B (Fig. 8C). Monensin-treated cells and cells ex- of H+-ATPases of the vacuolar system, on the secretory

19098 Inhibitory Effects of Concanamyein B

Intact Cells Permeabilized Cells

Concanamycin B GT45 + - + - Chase (min) 6 0 1 2 3 6 0 30 0 6 0 1 2 0 30 0 120 60 30 0 120 60 30 0

B

FIG. 6. N-Linked glycan modifications are incomplete in intact and permeabilized HepG2 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 of 1.0 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.

I Intact Cells I Permeabilized Cells I ~ ~ _ _ _ _ _ _

Concanamycin B + - - I + I I

Chase (min) 6 0 1 2 0 6 0 30 0 6 0 1 2 0 0 30 123 60 30 0 120 60 30 0

A

0.

Sialic Acids

1%

- ”” -= - ”- “ ” ” ”

1

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 pre- vents 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 implies that more than one pathway exists for TGN to PM trafficking for soluble

A control

Inhibitory Effects of Concanamycin B

B 15 min concanamycin B C

19099

90 min concanamycin B

D 90 min brefeldin A E 15 min monensin F 90 min monensin

FIG. 8. Morphology of the Golgi apparatus is slightly altered after exposure to concanamycin B. HepG2 cells treated as indicated were prepared for immunofluorescence as described under “Experimental Procedures.” Golgi morphology was analyzed with either anti-a- mannosidase 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 V- ATPases can result in protein missorting along the vacuolar system (Yamashiro et al., 1990; Klionsky et al., 1992). These reports have provided the strongest evidence so far that vacuolar ATPases play a role in efficient and correct sorting of proteins to the yeast vacuole. Delivery to the vacuole, which resembles the mammalian lysosome, is altered by protonop- hores, acidotropic agents, and by bafilomycin (Banta et al., 1988; Klionsky et al., 1990). It was proposed that 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 sialyltransfer- ases located in the trans-Golgi and TGN (Duncan and Korn- feld, 1988) may require 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 and Vassalli, 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 concana- mycin 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 com- plex-type glycan modifications, and of sialic acid transfer in particular, is far more pronounced indicating that permeabil- ization promotes access of concanamycin B to its target(s). Under these conditions, the majority of a1-AT is non-sialyl- ated. 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 inhib- ited. The reduction in complex-type N-linked glycan modifi-

19100 Inhibitory Effects of Concanamycin B cations, particularly sialylation, may stem from an effect of pH on activity of the transferases themselves, or on the availability of the nucleoside-sugar substrates. It is conceiv- able that the ApH in the tram-Golgi contributes to delivery of substrate or efflux of nucleoside phosphates (Hirschberg and Snider, 1987). Thus a dissipation of ApH could prove inhibitory in a less direct manner to reactions catalyzed by glycosyltransferases.

Most of the organelles of the vacuolar system contain H+- ATPases which are responsible for generating an internal acidic environment (Forgac, 1989). The use of inhibitors of V-ATPases such as concanamycin B and its structural rela- tives, the bafllomycins, is advantageous because of their es- tablished high selectivity. Unlike acidotropic agents such as primaquine, chloroquine, and ammonium chloride (Ohkuma and Poole, 1978; Strous et aL, 1985; Anderson and Pathak, 1986), the effecta of macrolide antibiotics can be assigned to specific enzymes. For this same reason, the macrolide antibi- otics with their effective concentrations in the nanomolar range are tools preferable over ionophores such as monensin. We observed that at least 10-fold higher concentrations of monensin are required to generate effects similar to those of concanamycin B (data not shown). Concanamycin B does appear to exert some pleiotropic effects on cellular structure. Analysis of Golgi morphology in concanamycin B-treated cells shows alterations that differ from those produced by monen- sin. Because vectorial transport is detected in the presence of concanamycin B, budding and fusion events must continue to occur. We have examined the effects of concanamycin B on the constitutive pathway in other cell systems. Preliminary studies of major histocompatibility complex processing in lymphoblastoid cells exposed to concanamycin B show a similar delay in surface deposition, and incomplete processing of N-linked glycans, of MHC Class I and I1 molecule^.^ The effects of concanamycin B therefore appear to be generaliza- ble. Secretion of IgM in plasma cells, however, was reported to be unaffected by chloroquine and ammonium chloride (Thorens and Vassalli, 1986). This suggests that the require- ments for pH may differ in various cell types and may depend on factors such as cell polarity.

Unlike yeast, mammalian mutants with defects in acidifi- cation (Laurie and Robbins, 1991) are not as readily obtained, and any observations are necessarily restricted to such mutant cell lines. Reagents such as concanamycin B which either neutralize low pH compartments or dissipate H+ gradients in a specific manner are therefore useful in dissecting the role of acidification in directing membrane traffic, or controlling

’ P. Benaroch, M. Yilla, and H. L. Ploegh, unpublished data.

post-translational modifications.

Acknowledgments-We thank Drs. Monty Krieger, Kelly More- men, and Steve Podos for generously providing reagents and Drs. Jeremey Auey, Monty Krieger, Harvey Lodish, Ton Schumacher, and Steve Podos for critically evaluating this manuscript and for their helpful discussions.

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