of biological chemistry val. 264, no. of 25, pp. 19956 … · 2017-11-13 · the journal of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 hy The American Society For Biochemistry and Mofecular Bioiogy, fnc. Val. 264, No. 33, Issue of November 25, pp. 19956-19966,1989

Printed in U. S A.

Relationship between Golgi Architecture and Glycoprotein Biosynthesis and Transport in Chinese Hamster Ovary Cells”

(Received for publication, June 15, 1989)

Nancy L. Stults, Marcus Fechheimer$g, and Richard D. CummingsOlI From the Departments of TBiochernistry and $Zoology, University of Georgia, A t k n s , Georgia 30602

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with 16-3Hlgalactose or [2-3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 NM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of t3H]mannose- and [3~]galacto~e-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid- treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine- linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.

The endoplasmic reticulum (ER)’ and Golgi apparatus play central roles in the biosynthesis and trafficking of secretory,

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* This work was supported by a Bioresources and Biotechnology grant from the University of Georgia (to R. D. C . and M. F.) and National Institutes of Health Grants CA37626 (to R. D. C.), GM35428, and BRSG S07RR07025-23 (to M. F.). Portions of this work were presented at the joint ASCB/ASBMB meeting in San Francisco, CA in January, 1989 (Stults et al., 1988). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. The abbreviations used are: ER, endoplasmic reticulum; Asn,

asparagine; CB-NBD-ceramide, N-[7-(4-nitrobenzoxa-1,3-diazole)]-6- aminocaproyl sphingosine; CHO, Chinese hamster ovary; Con A, concanavalin A DiOCs(3), 3,3‘-dihexyloxac~rbocyanine; HBSS, Hank’s balanced salt solution; RCA I, Ricinus communis agglutinin type I; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; Me2S04, dimethyl sulfoxide.

lysosomal, and integral plasma membrane proteins (Palade, 1975; Farquhar and Palade, 1981; Farquhar, 1985; Roth, 1987). The ER is a continuous cisternal and tubular membra- nous network that extends outward into the cytoplasm from the cell center. During interphase, the Golgi apparatus has a compact morphology and a perinuclear location consisting of stacks of cisternae that possess biochemical and topological polarity. Newly synthesized proteins are transported vectori- ally via vesicles from the rough endoplasmic reticulum to the cis, medial, and trans Golgi compartments. In the last major compartment, the trans Golgi network, proteins are sorted into different vesicles and sent to their final cellular destina- tions (Farquhar, 1985; Griffiths and Simons, 1986). As newly synthesized proteins pass through the ER and Golgi apparatus, they undergo a variety of co- and post-translational modifications including proteolytic cleavage, phosphorylation, sulfation, and glycosylation. With respect to glycosylation, the membrane organization of the ER and Golgi is thought to facilitate the sequential addition and/or trimming of sugars by compartmentalizing the various glycosidases and glycosyltransferases invoived in these reactions (Dunphy and Rothman, 1985; Kornfeld and Kornfeld, 1985; Lennarz, 1987; Roth, 1987).

Since the morphology of the ER and Golgi apparatus is likely to be of functional significance, insight into the mechanisms responsible for their biogenesis and maintenance is currently being sought. Based on a variety of biochemical and morphological evidence, the organization of these membrane- bound organelles appears to be dependent on the microtubul~ network. Similarities in the distribution of microtubules and ER membranes have been demonstrated (Terasaki et al., 1984, 1986). In addition, ER membranes have been reported to bind coichicine (Riordan and Alon, 1977) and to inhibit microtubule polymerization (Reaven and Azhar, 1981). Microtubule- depolymerizing drugs including nocodozole, colcemid, and colchicine reversibly alter the extended lattice-like morphology of the ER by causing it to retract toward the cell center (Louvard et al., 1982; Terasaki et al., 1984, 1986).

There is a strong body of evidence indicating that microtubules direct the organization of the Golgi apparatus. For example, 1) the Golgi apparatus and the microtubule organizing center of interphase cells are proximal to one another (Kupfer et al., 1982; Rogalski and Singer, 1984); 2) during mitosis both the microtubule organizing center and the Golgi apparatus are dispersed (reviewed by Thyberg and Moska- lewski, 1985); 3) disassembly of microtubules with drugs such as colchicine, colcemid, or nocodozoie is accompanied by fragmentation of the Golgi apparatus which is reversible upon removal of the drug (for review see Thyberg and Moskalewski, 1985); and 4) perturbations of the microtubular network by antibodies, taxol, and nonhy~olyzable analogs of GTP cause concomitant rearrangements of the Golgi apparatus (Wehland et al., 1983, Wehland and Sandoval, 1983). Together, these

19956

Role of Golgi Ar~hitecture in ~lycoprotein Biosynthesis and Transport 19957

observations suggest that there are physical contacts between the cisternae of the Golgi apparatus and cytoplasmic microtubules. In this regard, it has been shown that isolated Golgi membranes bind to microtubules (Reaven and Azhar, 1981), and a microtubule binding protein of 110 kDa, which may be involved in such contact points, has been isolated from the cytoplasmic face of Golgi membranes (Allan and Kreis, 1986). However, the functional significance of the interactions of microtubules with the ER and Golgi apparatus remains to be established.

In addition to providing structural support for the cell and its organelles, microtubules direct the cytoplasmic transport of vesicles and certain organelles (reviewed by Vale, 1987). Portions of the receptor-mediated endocytic pathway (Ostlund et al., 1979; Kusiak et at., 1980; Wolkoff et ai., 1984) as well as a number of Golgi-mediated sorting events also appear to be facilitated by microtubules. For example, microtubules have also been implicated in both constitutive and regulated secretion, and there is evidence that suggests their involvement in the establishment of cell polarity and polarized exocytotic events (reviewed by Kelly, 1985; Burgess and Kelly, 1987; Vale, 1987). In view of the apparent role of the microtubule network in the organization of the ER and Golgi apparatus and its involvement in vesicular transport, it was of interest, to investigate the role of microtubules in the biosynthetic pathway of endogenous cellular glycoproteins in a nonpolarized cell such as the Chinese hamster ovary (CHO) cell.

Previous studies have shown that anti-microtubular drugs do not cause any gross alterations in the incorporation of specific sugars into certain serum (Banerjee et al., 1976), membrane (Quaroni et al., 1979; Blok et al., 1981; Bennett et al., 1984a, 1984b), or viral (Rogalski et al., 1984) glycoproteins. We reasoned that the effect of anti-microtubular drugs on glycoprotein biosynthesis might be much more subtle than the simple inhibition of monosaccharide inco~oration. The sequence and linkage positions of monosaccharides compris- ing oligosaccharide chains could be very different in cells in which the morphology of the ER and Golgi apparatus are radically altered. Using the technique of metabolic radiola- beling combined with lectin affinity chromatography, ion exchange chromatography, and methylation analysis, the fine structure of asparagine (Asn)-linked oligosaccharide chains on glycopeptides isolated from CHO cells treated with colcemid was examined (Cummings et aE., 1989). We found that colcemid-treated CHO cells efficiently synthesized glycoproteins with oligosaccharide structures indistinguishable from those synthesized by cells having a morphologically intact ER and Golgi apparatus. In addition, colcemid treatment had no detectable effect on the transport of newly synthesized glycoproteins to the cell surface. These results suggest that microtubules are not required for inter- or intra-Golgi trafficking during glycoprotein biosynthesis and do not facilitate the movement of vesicles containing integral membrane proteins from the Golgi to the plasma membrane in CHO cells.

EXPERIMENTAL PROCEDURES

Materials-The following reagents were obtained from the indicated sources: aprotinin, bovine serum albumin (BSA), colcemid, dibutyl phthalate, goat anti-mouse IgG-fluorescein isothiocyanate conjugate, leupeptin, pepstatin, p-phenylenediamine, phenylmethylsulfonyl fluoride, and QAE-Sephadex from Sigma; [6-3H]galactose and [2-3H]mannose from ICN Biomedical; Eagle’s a-minimal essential medium, fetal calf serum, glutamine, penicillin, streptomycin, trypsin, and Hank’s balanced salt solution (HBSS) from Flow Labs; concanavalin A (Con A)-Sepharose from Pharmacia LKB Biotech- nology Inc.; Ricinus communis agglutinin I (RCA &agarose from E- Y Labs; Pronase from Calbiochem; N-[7-(4-nitrobenzo-2-oxa-1,3-

diazole)]-6-aminocaproyl sphingosine (C~-NBD-ceramide) and 3 3 - dihexyloxacarbocyanine (DiOC&)) from Molecular Probes; Apiezon A from Biddle Instruments (Bluebell, PA); and protein standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) from Bio-Rad. The mouse monoclonal antibody (DMlA) against a-tubulin (Blose et al., 1984) was a generous gift from Dr. Charles Keith, University of Georgia. Tomato lectin-Sepharose was prepared by coupling to CNBr-activated Sepharose 4 8 (Sigma) as described previously (Merkle and Cummings, 1987a, 1987b). N,N’,N”-Triacetylchitotriose and N,N’,N”,N”’-tetraacetylchitotetraose were prepared from chitin (Sigma) by the procedure of Rupley (1964).

Cell Culture and Treatment of CHO Cells with Colcemid-CHO cells were cultured routinely in 100-mm tissue culture plastic dishes (Falcon) in a-minimal essential medium containing 10% fetal calf serum and 100 IU/ml penicillin, 100 pg/ml streptomycin, and 2 mM glutamine. Cells were exposed to 10 p~ colcemid in complete medium for 3 h at 37 ‘C. Colcemid was added to the culture medium from a stock solution of 10 mM in Me,SO,. Control cells were incubated similarly in the presence of 0.1% Me2S04.

Fluorescence Staining-Immunofluorescence staining of microtubules of CHO cells was performed on cell monolayers grown on glass coverslips. After treatment of the cells with Me2S04 or colcemid, the cell medium was removed, and the cells were washed extensively with PBS (phosphate-buffered saline: 6.7 mM KH2P04, pH 7.4, 150 mM NaCl) and then fixed with 3.7% formaldehyde in PBS. After washing the cells with PBS containing 0.2 M glycine, the cells were permeabilized by exposure to 0.1% Triton X-100 for 5 min. The coverslips were incubated with a mouse monoclonal anti-a-tubulin antibody (DMlA) followed by fluorescein-labeled goat anti-mouse IgG in PBS containing 0.5% BSA for 30 min. The coverslips were washed with PBS containing 0.1% BSA between and following the antibody in- cubations. The coverslips were mounted on glass slides with 90% glycerol in PBS containing 1 mM p-phenylenediamine. The slides were viewed with a Zeiss IM 35 microscope equipped with differential interference contrast optics and for epifluorescence. Photographs were taken using a Zeiss 63X planapo objective (N.A. 1.4) with an Olympus OM-4 camera using Kodak Tri-X 400 ASA film.

The Golgi apparatus of Me2S04- and colcemid-treated cells grown in microscope chambers was stained with the fluorescent sphingolipid Cs-NBD-ceramide as described previously (Lipsky and Pagano, 1985). Briefly, the lipid (10 nmol/ml) was administered to the cells in culture medium containing 10 mM Hepes and 0.68 mg/ml BSA. The cells were incubated at 37 “C for 10 min after which the medium was removed and the cells washed with HBSS. Fresh culture medium at room temperature was added to the chambers, and the cells were photographed as above using the fluorescein filters. The ER of celfs treated with Me2S04 or colcemid was visualized by staining with the lipophilic cationic dye DiOCs (3) as described previously (Terasaki et al., 1986).

Pulse Labeling of CHO Cells with Radiolabeled Sugars-Cells at 60- 80% confluency in 60-mm tissue culture dishes were pulse-labeled for 20 min with 20-50 pCi/ml [6-3H]galactose or [2-3H]mannose at 37 ‘C in the presence of 0.1% Me2S0, or 10 p~ colcemid in Me2S04. After the pulse, the medium was removed, the cells washed with HBSS, and fresh medium added to commence the chase. At each chase time, the medium was removed and the cells were washed 3 to 4 times with 2 ml of ice-cold HBSS or PBS. The cells were harvested by scraping them from the plates with a rubber policeman in 1 ml of HBSS or PBS and then lyophilized prior to further analysis.

Preparation of Radiolabeled Glycopeptides-The lyophilized radiolabeled cell pellets were extracted with 1 ml of CHCl~:CH30H(2:l~ followed by 1 ml of CHCb:C&OH:H20 (10:10:3) to remove lipids. Alternatively, the cell suspensions were sonicated briefly and then extracted with 10 volumes each of CHCl,:CH,OH and CHC1a:CH30HH20 as above. The extracted pellets were allowed to air dry and were then digested with 5 mg/ml Pronase in 0.1 M Tris- HCl, pH 8.0, containing 1 mM each of CaC12 and MgC12 at 60 “C for 18-24 h as described (Cummings et al., 1989). The resulting Pronase digests were desalted over Sephadex (2-25 in 7% n-propyl alcohol in water, and the radiolabeled glycopeptides were recovered in the void volume.

Preparation of Radiolabeled Glycoproteins and SDS-PAGE-A por- tion of the cell sonicate from pulse-labeled CHO cells was added to an equal volume of PBS containing 0.5 mM phenylmethylsulfonyl fluoride, 1 pg/ml pepstatin, 5 pg/ml aprotinin, 10 pg/ml leupeptin, and 3.4 mM EDTA. The proteins were precipitated on ice by addition of phosphotungstic acid and trichloroacetic acid for a final concen-

19958 Role of Golgi Architecture in Glycoprotein Biosynthesis and Transport

tration of 5% phosphotungstic acid, 10% trichloroacetic acid. The resulting pellets were washed with cold acidified acetone (0.2% HCI) followed by acetone (1 ml each wash}. The pellets were then solubilized in 0.1 N NaOH, suspended in sample buffer, boiled, and loaded onto a 6-18% gradient polyacrylamide slab gel for one-dimensional electrophoresis in SDS under reducing conditions (Laemmli, 1970). Apparent M, values were estimated by using a standard mixture of myosin (ZOO,OOO), @-galactosidase (116,250), phosphorylase b (92,500), BSA (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soy- bean trypsin inhibitor (22,500), and lysozyme (14,400). The radiolabeled bands were processed for fluorography with EN3HANCE (Du Pont-New England Nuclear). The gel was dried and exposed to Kodak X-Omat AR film at -80 "6. To determine the incorporation of radiolabeled sugar into total cell glycoproteins, aliquots of the solubilized phosphotungstic acidftrichloroacetic acid precipitates were assayed for protein content (Lowry et al., 1951) using BSA as the standard and for radioactivity using a Beckman LS 1801 liquid scintillation counter.

Lectin Affinity Chromatography of ~ ~ ~ o l ~ e l e d Glycopeptides- Lectin affinity chromatography of radiolabeled glycopeptides on columns (1-2-ml bed volume) of Con A-Sepharose, RCA I-agarose, and tomato lectin-Sepharose was carried out at room temperature as described previously (Merkle and Cummings, 1987a). Fractions (1-2 ml) were collected and monitored for radioactivity by liquid scintillation counting. Fractions containing bound and unbound radioactivity were pooled and concentrated by evaporation on a rotary shaker evaporator.

Column Chromatography-Glycopeptides were desalted and freed from sugar haptens by Sephadex G-25 chromatography in 7% n- propyl alcohol in water. QAE-Sephadex chromatography was carried out using 1-mi columns by batch elution with increasing concentrations of NaCl in 2 mM Tris base (Varki and Kornfeld, 1980).

Desialylation and Methylation Analysis of Glycopeptides-Radiola- beied glycopeptides were desialylated by treatment with 2 N acetic acid at 100 "C for 1 h. Acetic acid was removed by evaporation under reduced pressure. (3H]Galactose-labeled glycopeptides were methylated using a modification (Gunnarson, 1987) of the iodo- methane:NaOH:dimethyl sulfoxide procedure (Ciucanu and Kerek, 1984). The permethylated oligosaccharides were hydrolyzed in 2 N trifluoroacetic acid for 4 h a t 100 "C, cooled, and evaporated under Nz. The methylated galactose species were separated by thin layer chromatography on Silica Gel G in the solvent system acetone:water:ammonium hydroxide (250:3:1.5) (Stoffyn et at., 1971). The sample lanes were scraped in 0.5-cm sections and radioactivity determined by liquid scintillation counting. The partially methylated monosaccharides were identified by cochromatography with authentic standards as described previously (Cummings and Kornfeld, 1984).

Trypsin Retease of Cell Surface-~sociated R~dioaetivi~-CHO cells (60-mm dishes) were pulse-labeled with 100 pCi/ml [6-3H]galactose for 20 min at 37 "C in culture medium containing 0.1% Me2S04 or 10 p~ colcemid as described above. Trypsin treatment of the labeled cells harvested at different chase times was carried out in 1 ml of HBSS containing 2.5% trypsin and 2 mM EDTA at 10 "C for 10 min. The cells were completely detached from the plate and were greater than 90% viable based on the exclusion of trypan blue. To terminate the digestion, the cell suspensions were centrifuged over a mixture of Apiezon Adibutyl phthalate (k9) to separate the cells from the buffer. The resulting trypsin supernatant solutions and cell pellets were digested with 10 mg/ml Pronase as described above. The resulting Pronase digests were desialylated and passed over RCA I-agarose to assess trypsin-released cell surface and cell-associated glycopeptides having terminal pl,4-Iinked galactose residues.

RESULTS

Effect of Colcemid on the Morphology of the ER and Golgi Apparatus in CHO Cells-The effect of microtubule disruption on the morphology of the ER and Golgi apparatus in CHO cells was examined after treatment of the cells with 10 pM colcemid in complete medium for 3 h at 37 "C. The cells were processed for immunofluore~ence staining of tubulin using a mouse monoclonal anti-a-tubulin antibody, for vital staining of the Golgi apparatus with the fluorescent sphingolipid, Cg- NBD-ceramide, or for labeling of the ER using the lipophilic fluorescent dye, DiOC,(3). Control cells that were treated with 0.1% MezSOa (Fig. la) exhibited a typical interphase

distribution of microtubules. In contrast, colcemid-treated cells (Fig. lb) revealed diffuse cytoplasmic staining indicating the disrupted state of the microtubules. The Golgi apparatus in coicemid-treated cells (Fig. Id) exhibited a punctate ap- pearance found throughout the cytoplasm, unlike control cells (Fig. IC) in which the Golgi had a well defined, compact perinuclear location. While the endoplasmic reticulum in control cells had an extended reticular network (Fig. le), in the presence of colcemid it was retracted from the cell periph- ery, and its lattice-like reticular morphology was altered (Fig. If). These results indicate, under the conditions of the colcemid treatment, that disassembly of microtubules in CHO cells is accompanied by fragmentation of the Golgi apparatus and disruption of the ER.

Effect of Colcemid on the I ~ o r ~ r ~ t i o n of p H ] G ~ c ~ o s e into CHO Cell Glycoproteins-The incorporation of [3H]galactose into total CHO cell glycoproteins pulse-labeled in the presence of 0.1% Me2S04 or 10 p~ colcemid was examined (Fig. 2). After labeling for 20 min, the cells were incubated in medium without radioactive sugar for up to 150 min. At the end of each chase time, the cells were washed and harvested from the plates. A cell sonicate was prepared, and the proteins were precipitated with 5% phosphotungstic acid, 10% trichloroacetic acid. The time course of incorporation of label over the chase period into acid-precipitable proteins in colcemid- treated cells paralleled that of control cells. Both curves reveal an apparent increase in [3H]galactose incorporation after 30 min of chase followed by a gradual decrease over the remain- der of the time course to approximately 60% of that seen at the end of the 20-min pulse period. The increase in ['HI galactose incorporation observed early in the chase period presumably reflects additional transfer of the label from the sugar nucleotide pool into glycoprotein oligosaccharide chains. Since [6-3H]galactose will also radiolabel glucose, the decreased incorporation seen at later chase times may be due to trimming of precursor chains (i.e. GlcaMangGlcNAczAsn). In the case of the colcemid-treated cells, approximately 30% less radioactivity was protein-associated at the end of the 20- min pulse period. This apparent inhibition in the incorporation of [3H]gaiactose into precipitable protein was maintained over the entire chase period.

Glycoproteins labeled with [3H]galactose for 20 min in an analogous pulse-chase experiment were analyzed by SDS- PAGE and fluorography (Fig. 3). Numerous glycoproteins were labeled with the majority having molecular masses in the range of 70-120 kDa. The pattern of labeling exhibited by colcemid-treated cells is indistinguishable from control cells at each chase time examined. In addition, there was no apparent change in the labeling pattern as a function of chase time, which indicates that the [3H]galactose-labeled glycoproteins are terminally processed and are not degraded or secreted to any significant extent during the chase period.

Effect of Colcemid on the Biosynthesis of Glycoprotein oligosaccharide Chains-To examine the effect of microtubule disassembly on the biosynthesis and processing of glycoprotein Oligosaccharide chains, glycopeptides isolated from CHO cells that had been treated with 0.1% Me2S04 or 10 pM colcemid and pulse-labeled with radiolabeled sugar were subjected to lectin affinity chromatography, ion exchange chromatography, and methylation analysis. The cells were labeled with 2-[3H]mannose or 6-[3H]galactose for 20 min, washed, and then incubated in medium without radioactive sugar for varying periods of time. At the end of each chase time, the cells were washed with ice-cold PBS and harvested from the plates. The resulting cell pellets were extracted with chloro- form and methanol to remove glycolipids and then digested

Role of Golgi Architecture in Glycoprotein Biosynthesis and Transport 19959

FIG. 1. Disassembly of microtubules in CHO cells is accompanied by dispersal of the Golgi apparatus. CHO cells were incubated at 37 "C in medium containing 0.1% Me2S0, (a, c, e) or 10 p~ colcemid (b, d, f ) for 3 h. Microtubules were visualized in fixed, permeabilized cells by immunofluorescence labeling with a mouse anti-a-tubulin antibody followed by a fluorescein-labeled goat anti-mouse IgG (a, b). The Golgi apparatus was stained in living cells using C6-NBD-ceramide (c, d). The ER was labeled in fixed cells using the fluorescent dye, DiOCd3) (e, f). Bar, 10 pm. Arrowheads denote the cell margin. The microtubules, Golgi, and ER were most readily perceived in cells that were extensively spread out on the substrate. Considerable out-of-focus fluorescence was observed in more rounded cells as seen in panel a.

19960 Role of Golgi Architecture in Glycoprotein Biosynthesis and Transport 300, 1

8 F I

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0 30 6 0 90 120 150

CHASE TIME (MIN)

FIG. 2. Effect of colcemid on the incorporation of ['Hlgalac- tose into total cell glycoproteins. CHO cells were pulse-labeled for 20 min with [3H]galactose and then chased for different periods of time in medium without radiolabeled sugar in the presence of either 0.1% Me2S04 (DMSO) or 10 PM colcemid. At the end of each chase time, the medium was removed and the cells were washed, harvested and sonicated in ice-cold PBS. After addition of protease inhibitors, the proteins were precipitated with 5% phosphotungstic acid, 10% trichloroacetic acid. The resulting acid-insoluble pellets from Me2S04- (m) and colcemid (0)-treated cells were solubilized in 0.1 PI NaOH and assayed for protein content and radioactivity.

Chase 0 30 60 90 150

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116- 93-

66-

45-

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22- 14-

FIG. 3. SDS-PAGE of [sH]gdactose-la~led proteins from Me,SO,- and colcemid-treated CHO cells. CHO cells were pulse- labeled for 20 min with [3H]galactose and chased for different periods of time in medium without radioactive sugar in the presence of 0.1% Me2S04 or 10 PM colcemid. At the end of each chase time, the medium was removed and the cells were washed, harvested, and sonicated in ice-cold PBS. After addition of protease inhibitors, the proteins were precipitated with 5% phosphotungstic acid, 10% trichloroacetic acid. The resultingpellets from Me2S04- (-) and colcemid (+)-treatedcells were subjected to SDS-PAGE and fluorography. At the indicated chase times (rnin), the same amount of radioactivity (17,000-35,000 cpm) was loaded in control and experimental lanes. The positions of M, standards (X are indicated.

with Pronase to prepare [3H]galactose- and [3H]mannose- labeled glycopeptides. After desalting the Pronase digests on Sephadex G-25, the glycopeptides were fractionated on Con A-Sepharose to obtain a fingerprint of the relative amounts of the different types of oligosaccharide chains present (Cum- mings and Kornfeld, 1982; Merkle and Cummings, 1987a). The triantennary and tetraantennary complex-type Asn- linked oligosaccharides and 0-linked oligosaccharides, which do not bind to Con A, are found in the unbound fractions

designated as the Con A I pool. Glycopeptides eluted by 10 mM a-methyl mannoside (Con A 11) consist of complex-type biantennary Asn-linked chains. High mannose- and certain hybrid type Asn-linked oligosaccharides that bind to Con A with higher affinity (Con A 111) require higher concentrations of a-methyl glucoside or a-methyl mannoside for elution.

The Con A-Sepharose elution profiles of [3H]mannose- labeled CHO cell glycopeptides derived from control and colcemid-treated cells are shown as a function of chase time in Fig. 4. There was no significant alteration in the distribution of [3H]mannose glycopeptides in the three Con A pools as a result of colcemid treatment. The processing of high mannose-type to complex-type chains was apparent over the 90-min time course as evidenced by the increasing size of the Con A I pool. The [3H]galactose-labeled glycopeptides derived from control and colcemid-treated cells exhibited similar fractionation patterns on Con A-Sepharose (data not shown) indicating that core glycosylation was not significantly altered by colcemid. However, in contrast to the [3H]mannose label, the percentage of [3H]galactose radioactivity in the Con A I pools did not increase significantly over the 150-min chase period. This result is expected since transfer of galactose occurs at a more terminal step in the glycosylation pathway.

To investigate more subtle colcemid-induced perturbation of galactosylation, the [3H]mannose- and [3H]galactose-labeled Con A I glycopeptides were fractionated on RCA I- agarose that binds terminal /31,4-linked galactose residues (Merkle and Cummings, 1987a). When the [3H]mannose- labeled Con A I glycopeptide pools shown in Fig. 4 from control and colcemid-treated cells were applied to RCA I- agarose, the proportion of radioactivity eluted with 0.1 M lactose was found to be similar (data not shown). In addition, there was a chase time-dependent increase in the amount of glycopeptides bound to RCA I-agarose, indicating increasing levels of terminal glycosylation with galactose. The [3H]galactose-labeled Con A I glycopeptides obtained after 150 min of chase from control and colcemid-treated cells were also fractionated on RCA I-agarose. Approximately 30% of the radioactivity was bound and eluted with 0.1 M lactose (Table I). In each case, removal of sialic acid by mild acid treatment prior to the chromatography resulted in roughly a 2-fold increase in the amount of RCA I-bound radioactivity indicating the exposure of terminal /31,4-linked galactose residues.

Portions of the [3H]galactose-labeled Con A I glycopeptide pools isolated after 150 min of chase were fractionated on columns of QAE-Sephadex before and after desialylation with mild acid (Fig. 5). Negatively charged glycopeptides that bound to the column were eluted with a step gradient of NaCl in 2 mM Tris. Glycopeptides derived from Me2S04-treated (Fig. 5A) and colcemid-treated (Fig. 5B) cells exhibited very similar elution profiles. Less than 20% of the radioactivity was neutral while the remaining glycopeptides carried multiple negative charges. In each case (Fig. 5, C and D), after desialylation, approximately 50% of the radioactivity was no longer bound to QAE-Sephadex indicating the removal of sialic acid residues from the glycopeptides. The finding that a significant amount of the glycopeptides is still bound by QAE-Sephadex following desialylation can be attributed to negative charges associated with the peptide or sulfate groups on N-linked oligosaccharides (Roux et al., 1988). Thus, the fractionation patterns of native and desialylated Con A I glycopeptides obtained on RCA I-agarose and QAE-Sephadex reflect no significant effect of colcemid on galactosylation or sialylation of CHO cell complex-type oligosaccharide chains.

Methylation analysis was carried out on [3H]galactose- labeled Con A I glycopeptides obtained after 30 min of chase


100 1 00

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CHASE TIME (MIN)

FIG. 4. Effect of colcemid on the binding of [3H]mannose-labeled CHO cell glycopeptides to Con A- Sepharose as a function of chase time. [3H]Mannose-labeled glycopeptides were prepared from CHO cells that had been pulse-labeled for 20 min and chased for the indicated periods of time in medium without radioactive sugar in the presence of 0.1% MezS04 or 10 KM colcemid. The glycopeptides were chromatographed on Con A- Sepharose and the fractions were monitored for radioactivity. Unbound glycopeptides are designated Con A I. Glycopeptides eluted with 10 mM a-methyl glucoside are designated Con A 11. Glycopeptides eluted by 500 mM a- methyl glucoside and 500 mM a-methyl mannoside are designated Con A 111. The distribution of radioactivity in the different Con A pools at the different chase times are shown for MezS04-(solid) and colcemid (hatched)-treated cells.

TABLE I Effect of colcemid treatment on the binding of 13Hlgalactose-labeled

CHO cell Con A I glycopeptides to RCA I-agarose [3H]Galactose-labeled Con A I glycopeptides were prepared from

CHO cells that had been pulse-labeled for 20 min and then chased in medium without radiolabeled sugar for 150 min in the presence of 0.1% MezSO, or 10 PM colcemid. Glycopeptides were applied to RCA I-agarose before and after desialylation with mild acid as described under “Experimental Procedures.” Bound radioactivity was eluted from the column with 0.1 M lactose.

Treatment Fraction Radioactivity

Bound Unbound

% MezS04 Con A I 34 66 Colcemid Con A I 33 67

MezSO, Con A I (-sialic) 58 42 Colcemid Con A I (-sialic) 64 36

in order to assess whether colcemid treatment of the cells resulted in any alterations in galactosyl linkage positions (Table 11). The methylated galactose species recovered in the glycopeptides indicated that the majority of the galactose residues are either substituted at the 3-position (-40%) or are unsubstituted (-40%). Removal of sialic acid by mild acid treatment results in about a 1.5-fold increase in the 2,3,4,6- tetramethyl derivative reflecting exposure of terminal galactose residues. The concomitant decrease in the amount of the 2,4,6-trimethyl derivative after desialylation demonstrates that sialic acid residues are linked to the 3-position of galactose. The 2,4,6-trimethyl species (-25%) remaining after desialylation can be attributed to pl,3-linked galactose residues contained in the poly-N-acetyllactosamine [3GalO1,4Glc- NAcPl] sequence (Li et al., 1980; Merkle and Cummings, 1988). CHO cells are known to synthesize N-linked oligosaccharides containing a2,3-linked sialic acid residues and to lack a2,6-sialic acid (Footnote 2; Sasaki et al., 1987; Takeuchi et al., 1988). The material (15-20%) that comigrated with 2,3,4-trimethylgalactose must be derived from another un- known substituent, perhaps from 0-linked chains that would also be present in the Con A I fraction.

Consistent with the methylation results was the observation, based on tomato lectin binding, that colcemid treatment had little effect on the biosynthesis of poly-N-acetyllactosamine-containing oligosaccharide chains. Glycopeptides pos-

R. D. Cummings, unpublished observations.

sessing 3 or more linear units of the repeating N-acetyllactosamine disaccharide bind with high affinity to tomato lectin (Merkle and Cummings, 1987b). Approximately half of the radioactivity in [3H]galacto~e-labeled Con A I glycopeptides obtained after 90 min of chase from control or colcemid- treated cells were bound by tomato lectin-Sepharose, requir- ing elution with 20 mg/ml of a mixture of N,N’,N”-triacetylchitotriose and N,N,N,N-tetraacetylchitotetraose (data not shown). In summary, these data indicate that colcemid- induced disassembly of microtubules and reorganization of the endoplasmic reticulum and Golgi apparatus have no detectable effect on the extent or nature of the glycosylation of complex type oligosaccharide chains in total CHO cell glycoproteins.

Role of Microtubules in the Translocation of Newly Synthe- sized Glycoproteins to the Cell Surface-To assess whether microtubule or Golgi integrity is required for the translocation of newly synthesized glycoproteins from the Golgi apparatus to the plasma membrane, the amount of trypsin-sensitive radioactivity a t the cell surface of [3H]galacto~e-labeled control and colcemid-treated CHO cells was monitored. After labeling for 30 min, the cells were washed to remove free [3H] galactose and then chased in the presence of nonradioactive medium for up to 2 h. At the end of different chase times the cells were incubated briefly with trypsin in HBSS a t 10 “C to minimize intracellular vesicular movement. The duration of trypsin treatment (10 min) was chosen to allow significant release of glycopeptides without any apparent loss in cell integrity. Those [3H]ga1actose-labeled glycoproteins that have reached the plasma membrane are expected to be accessible to tryptic digestion and to be released as glycopeptides into the buffer. To terminate the trypsin digestion and to effect separation of the buffer from the cells, the cell suspensions were centrifuged through a layer of oil. The released tryptic glycopeptides in the buffer and the cell pellet-associated glycoproteins were digested with Pronase and desialylated by mild acid treatment. The resulting Pronase digests were passed over RCA I-agarose to assess the levels of glycopeptides having terminal pl,4-linked galactose residues. If colcemid had inhibited the movement of newly synthesized glycoproteins to the cell surface, then less radioactivity would be expected to be sensitive to trypsin treatment and thus fewer counts would be bound by RCA I-agarose.

As shown in Fig. 6, at each chase time examined, the level of trypsin-sensitive radioactivity relative to the total cell-

19962 Role of Golgi Architecture in Glycoprotein Biosynthesis and Transport

FIG. 5. QAE-Sephadex chromatography of [SH]galactose-labeled Con A I glycopeptides from MenS04- and colcemid-treated CHO cells. Con A I glycopeptides were prepared from CHO cells that had been pulse-labeled for 20 min with [3H]galactose and chased in medium without radiolabeled sugar for 150 min in the presence of 0.1% Me2S04 (DMSO) or 10 p M colcemid. The glycopeptides were applied to QAE- Sephadex columns (2 ml) in 2 mM Tris Base and eluted stepwise with 20, 70, 140, 200, 250, and 1000 mM NaCl as indicated by the arrows. Fractions (1.5 ml) were collected and monitored for radioactivity. Panels A and B show the profile for glycopeptides derived from Me2S04- and colcemid-treated cells, re- spectively. In panels C and D, the glycopeptides were desialylated by mild acid treatment prior to the chromatography

100

200 80

60 150

40 i5 100

20 50

0 0 1 0 20 30 4 0 0 1 0 20 30 4 0

0

FRACTION FRACTION

TABLE I1 Distribution of radioactivity in methylated galactose residues

following methylation of [3H]galactose-labeled Con A I glycopeptides Con A I glycopeptides were prepared from CHO cells that had been

pulse-labeled for 20 min with [3H]galactose and chased in medium without radiolabeled sugar for 30 min in the presence of 0.1% Me2S04 or 10 p M colcemid. The glycopeptides were methylated before and after desialylation with mild acid. The results are expressed as the percent of total radioactivity recovered as methylated galactose species.

Treatment Fraction Methylated galactose species

2,3,4- 2,4,6- 2,3,4,6-

%

Me2S04 Con A I 19 44 37 Colcemid Con A I 17 41 42

Me2S04 Con A I (-sialic) 12 23 65 Colcemid Con A I (-sialic) 15 27 59

associated radioactivity was not affected by prior incubation of the cells with colcemid. At the end of the 20-min pulse, approximately 4% of the total cell-associated radioactivity was sensitive to trypsin treatment, while after 30 min of chase about 9% of the counts were now released from the cells. The level of trypsin sensitivity did not change during the remain- der of the chase period, indicating that movement of newly synthesized glycoproteins to the cell surface was accomplished within 30 min. These results indicate that, in the case of CHO cells, neither the disassembly of microtubules nor fragmentation of the Golgi apparatus inhibits the transport of newly synthesized glycoproteins to the cell surface.

DISCUSSION

In order to state unequivocally that an organelle is functional under certain conditions of cell perturbation, it is essential to obtain detailed biochemical information regarding the reactions carried out by that organelle. Since a morphological feature of the ER and Golgi involves the compartmen-

0 3 0 6 0 120

CHASE TIME (MIN)

FIG. 6. Effect of colcemid on the translocation of newly synthesized glycoproteins to the cell surface. CHO cells were pulse-labeled for 30 min with [3H]galactose and chased for different periods of time in medium without radioactive sugar in the presence of 0.1% Me2S04 or 10 p M colcemid. After each chase period, the cells were washed with ice-cold PBS and incubated at 10 "C with 2.5% trypsin in Hank's balanced salt solution containing 2 mM EDTA for 10 min. An aliquot of each cell suspension was centrifuged over Apiezon Adibutyl phthalate (1:9) to effect separation of the buffer from the cells. The amount of radioactivity in glycopeptides containing terminal @1,4-linked galactose residues was compared following mild acid hydrolysis in the trypsin supernatant solutions with that remaining associated with the cell pellets by chromatography on RCA I-agarose.

talization of enzymes involved in glycoconjugate biosynthesis, determination of the structures of oligosaccharide chains synthesized by these organelles is a sensitive measure of their status. We have examined glycoprotein oligosaccharide structures as a measure of the functionality of the ER and Golgi apparatus in CHO cells in which the morphology of these organelles has been radically altered by depolymerization of microtubules. The experiments presented here demonstrate


that reorganization of the ER and dispersal of the Golgi apparatus by treatment with colcemid (Fig. 1) does not com- promise Asn-linked glycosylation of glycoproteins or their transport to the plasma membrane in CHO cells.

Effect of Microtubule Disassembly on Glycoprotein Biosyn- thesis-Previous studies of the effects of anti-microtubular drugs on glycoprotein biosynthesis have indicated that the incorporation of monosaccharides into specific glycoproteins is not significantly altered. For example, it has been reported that although colchicine treatment inhibits the secretion of rat hepatic serum glycoproteins, it does not inhibit the addition of the terminal sugars galactose and sialic acid to those glycoproteins (Banerjee et al., 1976). Similarly, colchicine has shown little effect on the incorporation of radioactive fucose into small intestinal and hepatocyte membrane glycoproteins (Blok et al., 1981; Quaroni et al., 1979; Bennett et al., 1984a, 1984b). Examination of the biosynthesis of vesicular stoma- titis virus G protein in nocodozole-treated cells by SDS-PAGE analysis of immunoprecipitates revealed no alteration in the rate or extent of sialylation (Rogalski et al., 1984). The results presented here in this study are consistent with and extend such previous findings.

The pattern of individual CHO cell glycoproteins metabolically radiolabeled with [3H]galactose was not altered by colcemid treatment (Fig. 3) nor were the structures of the oligosaccharides chains on [3H]mannose- or [3H]galactose-labeled glycoproteins synthesized by colcemid-treated cells found to be aberrant. Core glycosylation (Fig. 4), galactosylation (Tables I and 11), and sialylation (Fig. 5 and Table 11) of Asn-linked oligosaccharide chains were found to proceed normally in cells that lacked an intact microtubular network. Colcemid was found to partially inhibit the incorporation of [3H]galactose into total CHO cell glycoproteins (Fig. 2). This observation may be due to an inhibitory effect of colcemid on protein synthesis that has been reported for secreted hepatic plasma proteins (Redman et al., 1981). Such inhibition of protein synthesis may be a consequence of the altered morphology of the ER in colcemid-treated cells. In addition, it has been shown that nucleoside transport across cell membranes is inhibited by colchicine (Mizel and Wilson, 1972) which might cause depletion of the sugar-nucleotide pool in the Golgi apparatus, thus resulting in decreased transfer of radiolabeled monosaccharides to oligosaccaride chains. At high concentrations, colchicine has been reported to inhibit glycosyltransferase activities (Mitranic et al., 1981). However, under the conditions of our experiments, there was no detectable effect of colcemid on CHO cell UDP-Gal:GlcNAc@l,4- galactosyltransferase activity (data not shown).

In summary, our data imply that disruption of the ER network by anti-microtubular drugs did not significantly ham- per the progression of newly synthesized proteins to the Golgi apparatus nor was the organization of the Golgi cisternae into compact, perinuclear stacks a requirement for efficient biosynthesis and processing of glycoproteins. If the contents of the Golgi cisternae were mixed as a result of fragmentation, it would be predicted, due to competition of glycosyltransferases and glycosidases, that newly synthesized oligosaccharide chains could have altered structures compared with those made by a morphologically intact Golgi. An example of such competition is that between N-acetylglucosaminyltransferase V and galactosyltransferase. Galactosylation of Asn-linked glycopeptides prior to the action of N-acetylglucosaminyltransferase V blocks all acceptor activity toward N-acetylglucosaminyltransferase V (Cummings et al., 1982). It would therefore be hypothesized that if both glycosyltransferases were in the same Golgi compartment, cells would be unable

to synthesize the appropriate multibranched oligosaccharide structures. Thus, it is likely that compartmentalization of the glycosylation machinery is not altered as a result of microtubule disassembly.

Role of Microtubular Network in Vesicle Tramlocation- Microtubule-based vesicular transport has been directly visualized in a number of systems including corneal keratocytes (Hayden et al., 1983) and squid axoplasm (Allen et al., 1985). In addition, microtubule-directed movements of chloroplasts (Menzel and Schliwa, 1986), pigment granules (Beckerle and Porter, 1983; McNiven and Porter, 1986), and endosomes and lysosomes have been documented (Herman and Albertini, 1984; Matteoni and Kreis, 1987). Translocating activities that are believed responsible for such movements have also been identified. For example, kinesin has been shown to drive the movement of organelles toward the distally oriented “plus” ends of microtubules (Vale et al., 1985a), while MAP 1C appears responsible for movement in the opposite direction (Vale et al., 1985b; Paschal and Vallee, 1987; Paschal et al., 1987).

In this context, it is significant that microtubules are not required for movement of vesicles to and through the Golgi apparatus during glycoprotein biosynthesis (Figs. 2-5, Tables I and 11). This finding suggests that they are transferred in the proper sequence to the appropriate compartment by another mechanism. In addition, our data indicate that the close apposition of Golgi cisternae was not required for efficient transport. Consistent with these results is the finding that transport between successive compartments of the Golgi apparatus can be reconstituted in a cell free system (Balch et al., 1984). Since in vitro transport was almost as efficient as in the intact cell, the authors proposed a receptor-mediated mechanism that is responsible for vectorial movement of vesicles from Golgi donor membranes to recipient cisternae. Several laboratories have been investigating the energy and cofactor requirements for transport and sorting of proteins in the ER and Golgi cisternae (for review see Pfeffer and Roth- man, 1987).

The role of microtubules in directing vesicular traffic between the Golgi apparatus and the plasma membrane is less clear-cut. Based primarily on pharmacological evidence, microtubules have been implicated in both constitutive and regulated secretion in many different tissues (for example, see Banerjee et al., 1976; Redman et al., 1981; Williams, 1981; Boyd et al., 1982; Busson-Mabillot et al., 1982; Oda and Ikehara, 1982; Bennett et al., 1984a; Wild and Bennett, 1984). These studies indicate, with a few exceptions (Malaisse-Lagae et al., 1979; Busson-Mabillot et al., 1982), that movement of secretory proteins between the ER and Golgi is not impaired. Rather, it is the exit of secretory vesicles from the Golgi that appears to be inhibited by anti-microtubular agents resulting in the accumulation of secretory products in Golgi-derived vesicles. In contrast, our data suggest that the translocation of newly synthesized endogenous plasma membrane glycoproteins to the cell surface is not altered by microtubule disassembly (Fig. 6). In agreement with our results, translocation of endogenous proteins to the plasma membrane in intestinal cells (Quaroni et al., 1979; Blok et al., 1981; Bennett et al., 1984b; Hasegawa et al., 1987; Eilers et al., 1989) and hepatocytes (Bennett et al., 1984b; Hubbard and Steiger, 1988) has been shown to proceed in the absence of microtubules. Several immunofluorescence studies have also concluded that transfer of newly synthesized viral glycoproteins to the plasma membrane is not inhibited in host cells in which microtubules were disassembled or radically rearranged (Rogalski et al., 1984; Salas et al., 1986; Rindler et al., 1987).

19964 Role of Golgi Architecture in Glycoprotein Biosynthesis a n d Transport

Although movement of proteins destined for the plasma membrane does not appear to be microtuble-dependent, there is compelling evidence that the establishment of cell polarity and polarized exocytosis requires an intact microtubular network. For example, disassembly of microtubules alters the polarized expression of endogenous membrane proteins in intestinal cells (Quaroni et al., 1979; Blok et al., 1981; Bennett et al., 198413; Hasegawa et al., 1987; Eilers et al., 1989) and hepatocytes (Bennett et al., 198413; Hubbard and Steiger, 1988) and the polarized budding of viral glycoproteins (Ro- galski et ai., 198% Rindler et ai., 1987) in host cells. However, there is a conflicting report based on viral glycoprotein sorting which concluded that microtubule disassembly has negligible effect on the biogenesis of cell surface polarity (Salas et a!., 1986). Interestingly, in the intestinal and hepatic cells, transport of proteins that reside in the apical domain of the plasma membrane appears to be more sensitive to microtubule depolymerizing drugs than that destined for the basolateral domain (Quaroni et al., 1979; Blok et al., 1981; Bennett et al., 198413; Hubbard and Steiger, 1988; Eilers et at., 1989). A similar effect has also been noted in viral glycoprotein sorting (Rindler et al., 1987) and in the secretion of lysosomal enzymes from an intestinal cell line (Eilers et ai., 1989). To- gether, these observations can be reconciled only by proposing a differential effect of microtubule inhibitors on the processes of polarized and nonpolarized secretion and plasma membrane biogenesis.

In summary, certain secretory and polarized exocytotic traffic requires an intact microtubular network while biosynthesis and translocation of proteins to the plasma membrane in nonpolarized cells are insensitive to microtubule disassembly. In the cases where microtubule-depolymerizing drugs do affect vesicular transport, the inhibition is incomplete, suggesting that not all vesicles are translocated along microtubules. Thus, microtubules do not appear to be an absolute requirement for vesicular movement but may serve to enhance the rate of transport, particularly over long distances, and to determine the site of delivery at the plasma membrane (Bur- gess and Kelly, 1987; Vale, 1987). Evidence for a possible role of actin microfilaments in vesicular transport has also re- cently been reported. For example, myosin f has been shown to be capable of mediating vesicular movements along microfilaments (Adams and Pollard, 1986) and the actin binding protein, caldesmon, which is regulated by calmodulin, binds to secretory granules from adrenal medulla (Burgoyne et al., 1986). In addition, actin filaments appear to be directly involved in the movement of particles and organelles in plant cells (Allen and Brown, 1988). In general, however, the effect of cytochalasins on secretion is not predictable since these drugs have been reported to have no effect (Redman et al., 1981) and to be either stimulatory or inhibitory (Stossel, 1981). In addition, microfilaments are probably not required for movement of newly synthesized proteins to the plasma membrane since cytochalasin D does not affect the polarized insertion or budding of viral glycoproteins in host cells (Ro- galski et d. , 1984; Salas et ut., 1986).

Based on the available data, cytoskeletal targeting of newly synthesized proteins is not sufficient to explain polarized or nonpolarized insertion of vesicles at the plasma membrane. As discussed by Burgess and Kelly (1987), it has been specu- fated that additional targeting mechanisms must be operative which may involve recognition of receptors or “fusion sites” by transport vesicles on the cytoplasmic face of the plasma membrane. However, it is difficult to reconcile vesicular movements without a facilitated transport mechanism since the viscosity of the cytoplasm and the extensive meshwork of the

cytoskeleton would be expected to place severe limitations on diffusion (Rebhun, 1972). Thus, continued research efforts in this area are required to define further the mechanisms for cytoplasmic vesicular translocation and targeting.

Significance of the Interaction of ~ ~ r o t ~ u ~ s with the ER and the Golgi Apparatus-Microtubules appear to be responsible for generating and maintaining the extended state of the ER network based on the observation that the extension of ER tubules and polymerization of microtubules occurs con- currently (Terasaki et al., 1986; Dabora and Sheetz, 1988; Lee and Chen, 1988). Our results indicate that the microtubule- dependent morphology of the ER is not required for the processing or transport of nascent glycoproteins destined for the Golgi apparatus. It is possible that the extended nature of the ER may be more important for its participation in calcium me~bolism, phosphoIipid biosynthesis, and/or drug detoxifi- cation.

The possible molecular basis and significance of Golgi- microtubule interactions are better characterized than in the case of the ER. Several mechanisms by which the Golgi apparatus might fragment in the presence of anti-microtubular drugs have been proposed. As discussed by Thyberg and Moskalewski (1985), the mo~hological evidence suggests that the Golgi is not randomly fragmented in the presence of such drugs, but is dispersed as many small intact stacks of cisternae. Therefore, microtubules are not required for the stacked arrangement of cisternae, but for their assembly into a larger complex. This idea has precedent in the fact that plant and insect salivary gland cells (Farquhar and Palade, 1981; Thy- berg and Moskalewski, 1985) as well as mitotic HeLa cells (Lucocq et al., 1987) have multiple stacks of Golgi dispersed throughout the cytoplasm. In addition, treatment of isolated Golgi complexes with anti-microtubular drugs does not alter their stacked arrangement (Cluett and Brown, 1988). An alternative possibility, based on electron microscopic visuali- zation of metal-impregnated Golgi elements, is that the Golgi apparatus is actually a continuous structure consisting of a single set of stacked cisternae (Rambourg et al., 1981). It has been speculated that this structure may simply unfold in the presence of anti-microtubular drugs that could explain the rapid recompaction of the fragmented Golgi elements upon their removal (Rogalski and Singer, 1984). In either scenario, the “connections” between individual stacks of cisternae, which may be provided by the binding of Golgi-specific proteins to microtubules, could be disrupted in cells where the microtubular network is rearranged. Interestingly, the re- cently characterized 110-kDa Golgi microtubule binding protein (Allan and Kreis, 1986) has been reported to undergo cell cycle-dependent phosphorylation in Xenopus eggs (Allan et al., 1988) suggesting that phosphorylation may be involved in the regulation of Golgi-microtubule interactions.

What then is the advantage of the Golgi apparatus in assuming a compact, stacked perinuclear arrangement if not to facilitate efficient transport of newly synthesized proteins through the glycosylation machinery? Although much work remains to be done to address the structure-function relationship of the Golgi apparatus, there is evidence suggesting that its morphology may be important during cell migration. The Golgi apparatus and the microtubule organizing center have been shown to orient themselves together in the direction of migration, suggesting that they coordinately direct the insertion of newly synthesized membranes via Golgi-derived vesicles into the leading edge of the cell (Kupfer et al., 1982; Bergmann et al., 1983).

It is becoming increasingly apparent that the interaction of cell organelles with microtubules is probably a general phe-


nomenon. The microtubular network is responsible for directing the cell cycle-dependent organization of membrane- bound organelles other than the Golgi apparatus and the ER. For example, the morphology and positioning of yeast vacu- oles (Guthrie and Wickner, 1988) and endosomes and lysosomes (Herman and Albertini, 1984; Matteoni and Kreis, 1987; Swanson et al., 1987) have been shown to be radically altered in cells where the microtubular network is perturbed. In this regard, it seems possible that “disruption” of the Golgi could result not from the dissolution of the connections between the Golgi elements but from the failure to direct recy- cling membrane to a single perinuclear location. Thus, the dynamic nature of Golgi traffic could be intimately related to the disruption of the Golgi observed upon microtubule depolymerization. Definition of the molecular basis and morphological and functional consequences of dynamic reciprocal interactions of the cytoskeleton with membrane-bound organelles remains a challenging area of cell biology.

Acknowledgment-We thank Dr. Roberta Merkle for technical advice during the course of this study and for critically reviewing this manuscript.

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