Intracellular transport of lymphoid surface glycoproteins

.I. Mol. Sol. (1981) 150, 525-535

Intracellular

Transport

of Lymphoid

Surface Glycoproteins

Role of the Golgi Complex A. TARTAKOFF, D. HOESSLI AND P. VASSALLI Department of Pathology University of Geneva, Faculty of Medicine 1211 Geneva 4, Su&zerland

(Received 30 January

1981)

The biosynthesis of the heavy chains of two membrane glycoproteins, identified as immunoglobulin M and histocompatibility antigens, has been studied in [ “Slmethionine pulse-chase experiments by one and two-dimensional gel electrophoresis. Terminal sugar addition results in marked shifts in gel mobility that are mainly due to sialic acid addition, since they are sensitive to neuraminidase. These shifts are prevented when the ionophore monensin is present during the chase incubation. We conclude that both membrane IgMt and H2 heavy chains normally pass through the Golgi subsite defined by monensin and acquire terminal sialic acid distal to this site. Analysis of surface-iodinated control and monensin-treated cells indicates that, in the presence of monensin, newly synthesized, incompletely glycosylated IgM and H2 are not transported to the cell surface. Thus these membrane proteins appear to follow the same intracellular pathway as secretory proteins.

1. Introduction The Golgi complex has been demonstrated to be an obligatory way-station in the intracellular transport of secretory proteins. A less extensive literature suggests that integral proteins of the plasma membrane and lysosomal enzymes also pass through the Golgi complex (Tartakoff, 1980). Although morphological, histochemical and biochemical studies of the Golgi complex indicate that it comprises several subcompartments (vesicle populations, proximal to distal cisternae), there is little information on the differential function of these subcompartments. Recently, the ionophore monensin has been shown to interrupt secretory protein intracellular transport at the level of the Golgi complex, and to cause its accumulation within grossly dilated Golgi cisternae (Tartakoff & Vassalli, 1978). In the case of murine immunoglobulin M plasma cells, the Ig whose secretion is severely impaired by monensin is incompletely glycosylated and lacks fucose, galactose and sialic acid (Tartakoff & Vassalli, 1979). Since it has been shown that complete inhibition of glycosylation of IgM heavy chains does not prevent its intracellular transport and secretion, and since the action of monensin t Abbreviations

used : IgM. immunoglobulin

0022-2836/81/240525-l

1 $02.00/O

M ; H2, major mouse histooompatibility 525

0 1981 Arademic

antigen

Press Inc. (London)

Ltd.

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D. HOESSLI

AND

P. VASSALLI

appears not to involve inhibition of sugar transferases, it has been concluded that monensin defines a Golgi sub-compartment, distal to the dilated Golgi cisternae that accumulate IgM, and proximal to the site(s) of terminal glycosylation (Tartakoff & Vassalli, 1979). Both the heavy chains of membrane IgM and histocompatibility antigens are known to be synthesized on the bound polysomes of the rough endoplasmic reticulum (Dobberstein et al., 1979; Owen et aZ., 1980; Wernet, et nl., 1973). The growing nascent polypeptide chains presumably acquire asparagine-linked core oligosaccharides from dolichol-linked donors as they traverse the rough endoplasmic reticulum membrane (Sefton, 1977: Struck 8: Lennarz, 1980). Subsequently. but prior to arrival at the cell surface, the oligosaccharides acquire the terminal sugars, including sialic acid (Dobberstein et al., 1979: Jones, 1977: Krangel et al., 1979 : Nathenson & Cullen, 1974: Owen et aZ.. 1980; Vassalli et al.. 1980). By analogy with the study of secretory glycoproteins and viral membrane glycoprotein biosynthesis, the addition of terminal sugars is presumed to occur in the Golgi complex (Leblond & Bennett, 1977 : Schachter & Roseman, 1980: Tartakoff, 1980). The present study addresses the question of whether these two types of membrane proteins reach the cell surface through the same Golgi-related as secretory proteins; i.e. whether monensin interrupts their intracellular

and terminal

pathway

transport

glycosylation.

2. Materials and Methods (a) Cells Mouse splenic lymphocytes were liberated by teasing, treated with 075% (w/v) NH&l in 20 mw-Tris . HCl (pH 7.4) to lyse red cells, sedimented on a buffered sucrose gradient to eliminated large cells (Ryser & Vassalli, 1974) and washed in HBSS (Hank’s basic salt solution (Flow Labs., Irvine, Scotland). P1798 Balb/c lymphoma cells (Litton Bionetics Inc., Kensington, Md) were transplanted intraperitoneally in Balb/c mice (Lampkin & Potter, 1958; Mathieson et al., 1978). The ascitic cell suspension was sedimented and washed in HBSS (b) (7ell culture and labeling For 15 min of [35S]methionine pulse-labeling, cells were incubated at 5 x 10’ cells/ml in Dulbecco’s modified Eagle’s medium lacking methionine and supplemented with 91 mCi [‘%]Met/ml at 37°C. After the pulse, cells were sedimented, washed in HBSS and lysed in 95% (v/v) Nonidet P-40 (NP-40; Shell Chemicals) in HBSS or xx-incubated for chase intervals in modified Eagle’s medium supplemented with 5% (v/v) fetal calf serum. After lysis, samples were centrifuged for 30 min at 100,000g before immunoprecipitation. Surface iodination was as described by Hoessli et al. (1980). Stocks of monensin were prepared in ethanol shortly before use. (c) Immunoprecipitation Lymphocyte Ig was recovered by sequential incubation of clarified lysates with rabbit anti-Ig antiserum and formalinized Staphylococci as described previously (Tartakoff & H2 immunoprecipitation employed Vassalli, 1979; Vassalli et al., 1980). Lymphoma an Robinson alloantiserum (CBA anti Balb/c) supplied by Dr P.

MEMBRANE

GLYCOPROTEIN

TRANSPORT

,527

Heidelberg) or rabbit anti-cell surface antibodies (Dcutscheskrebsforschungszentrum, (Hoessli rt a,l., 1980). A total of 10 ~1 of alloantiserum or 10 to 20 pg of rabbit antibodies were added to the lysate of 3 x lo6 to 8 x lo6 cells. After 30 min at 37”C, the immune complexes were collected with protein A-Sepharose or Staphylococci, respectively. Ig immunoprccipitates were washed with 0050/6 NP-40 in 915 M-N&I, 5 mM-EDTA, 50 m&rTris. HCI (pH 7.4). Washing of H2 immunoprecipitates included 2M-urea. (d) ,Veuraminidase (Vibrio cholerae) treatment Treatment of living cells was according to the method of Nicolson (1973), using 91 unit enzyme/IO’ cells per ml in pH 6 buffer at 37°C for 60 min. Alternatively, immunoprecipitates were treated with 0.1 unit of enzyme in 05 ml of pH 6 buffer at 37°C for 30 min. (e) 0~1 electrophoresis Onr-dimensional gel electrophoresis was performed on 100, to 15yA gradient gels (Tartakoff & Vassalli, 1979). Ig immunoprecipitatcs were reduced with /3-mercaptoethanol before loading. Two-dimensional gel electrophoresis was according to the method of O’Farrell (1975) and employed pH 3.5 to 10 ampholines and 10% (w/v) polyacrylamide gels. After rlectrophoresis, gels were fluorographed (Bonner & Laskey, 1974). (r) Material8

] ‘%]methionine was prepared according to Crawford & Gestcland (1973). Na’251 was purchased from New England Nuclear. The enzymes for iodination were obtained from Sigma Chem. Co., St. Louis, Neuraminidase (IUrio cholerae) was from Behringwerke, Marburg, Germany. Monensin was a gift from R. Hamill of Eli Lilly and Co., Indianapolis. Protein A-Sepharose was from Pharmacia. All other materials were of the highest available grade.

3. Results (a) IgM

heavy chain biosynthesis

Mouse spleen lymphocytes were pulse-labeled with [ 35S]methionine for 15 minutes, washed and either lysed or returned to non-radioactive chase medium in the absence or presence of 0.05 PM-monensin. At the end of the pulse, analysis of immunoprecipitated Ig by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by autoradiography showed heavy (PI) chains as a broad band or a closely spaced doublet (Fig. 1, lane A). Earlier studies (Vassalli et al., 1980) have shown this mixture to include a species destined for the cell surface (CL,,,) and one destined for secretion (t+). After two hours of chase, the cell lysates indeed contain two well-separated p bands (Fig. 1, lane B). It has been shown that the upper band has a distinct polypeptide structure (Jaton & Vassalli, 1980) and reasons: (1) it corresponds to fully glycosylated I-L,.,,chains for the following comigrates with surface-radioiodinated p chains in one and two-dimensional gels: (2) it is selectively removed by treatment of the intact chased cells with Pronase ; and (3) it is sensitive to the action of neuraminidase, which makes it corn&ate with its pulse-labeled precursor (Vassalli et al., 1980). The same studies have shown that the lower p band is destined for secretion as mature pentameric 19 S IgM molecules. When chase incubation is performed in the presence of monensin, two closely

528

A. TARTAKOFF.

A

0

D. HOESSLI

C

AND

D

P. VASSALLI

E

F

FIG. 1. One-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography of lymphocyte IgM. Lanes A to C, [ “Slmethionine biosynthetic label. At the end of a 15 min pulse-label (lane A), the heavy chains (H) are seen as a broad band. After a 2-h chase (lane B), a pair of bands is distinguished. The upper band (arrowhead) is c(,,,and comigrates with surface-iodinated pm. When @05 M-monensin is included during the chase (lane C). a pair of bands is seen but the upper one is not as retarded as in lane B. Lanes D to F, iodinated cells. Freshly prepared cells (lane D), cells incubated 4 h in control medium (lane E) or cells incubated 4 h in the presence of @05 PM-monensin (lane F) were iodinated. In all cases the heavy chains have the same mobility. In the gel illustrated, the light chains (L) migrated off the bottom.

spaced TVchains are seen (Fig. 1, lane C): the lower band has the same mobility as incompletely glycosylated pSchains present in controls, while the upper band is less retarded than in the control chase sample, suggesting that it represents TV,,,chains that fail to undergo complete glycosylation in monensin-treated cells. To investigate whether or not the transport of pm chains was impaired in the presence of monensin, i.e. whether the incompletely glycosylated pLmchains reach the cell surface, lymphocytes were incubated for four hours in the presence or

MEMBRANE

GLYCOPROTEIN

TRANSPORT

529

absence of monensin, and then radioiodinated. The gel mobility of iodinated TV chains of monensin-treated cells was indistinguishable from that of controls (Fig. 1, lanes 1.) to F). No iodinated TVchains with t.he mobility of the biosynthetically labeled /.A,,,chains of monensin-treated cells (Fig. 1, lane C) were detected. (b) HZ heavy chain biosynthesis (‘ells of Balb/c (H2d) T lymphoma P1798 were pulse-labeled with [35S]methionine for 15 minutes, washed and either lysed or returned to non-radioactive chase medium in the absence or presence of 1 PM-monensin. The cell lysates were immunoprecipitated with the alloantiserum or the rabbit antibody preparation rendered specific for cell surface determinants by adsorption-elution from fixed lymphoma cells (see Materials and Methods). The former reagent recognizes preferentially H2Dd molecules, whereas the latter reacts with both H2Dd and H2K” molecules and 10 to 15 other surface glycoproteins (Hoessli et aE., 1980). The two-dimensional mobility of H2 species is known to be complex and to depend upon the labeling protocol employed. In the case of H2Dd, for example, ,Jones (1977) has reported that after 15 minutes of biosynthetic labeling with [3581methionine, two major spots are seen. As a function of a subsequent chase interval, the more basic and apparently smaller of the two disappears, while the second spot and several other relatively more acidic spots become predominant. In our experiments employing an alloantiserum, we observe just such results (Fig. 2(a)

FIG. 2. Two-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography of alloantiserum immunoprecipitates. Cells were pulse-labeled for 15 min with [35R]methionine and lysed at once (a) or returned to chase medium for 2 h (b). In (a) two spots are seen (small arrows). By reference to Jones (1977) they are H2Dd species. Although complete data are lacking, it is probable that after briefer pulse labeling a single species would be seen. After the chase incubation (b) the more basic spot has disappeared and in its place is a family of three more acidic spots (arrowheads). When monensin is present during the chase (c) the dominant spots are as in (a); however, each of these original spots is accompanied by a more acidic and fainter partner (small arrows). In (b) and (c) the faint spots above H2D” may correspond to H2Kd. In the isoelectric focusing dimension the basic end of the gel is to the left. “A” is actin.

530

A. TARTAKOFF.

D. HOESSLI

AND

P. VASSALLI

and (b)). Furthermore, especially after the chase interval, several faint additional spots are visible. These correspond to the H2Kd species described by Jones after a comparable chase.. When monensin was present during the chase, the dominant spots observed are the same as after pulse-labeling (Fig. 2(c) versus Fig. 2(a)). Two faint colinear spots are also present, : one equidistant between the two pulse-labeled species, and one that is more acidic. There are also several very faint spots of slightly lower sodium dodecyl sulfate/polyacrylamide gel mobility. These may be H2K” species. The efficiency of immunoprecipitation was much greater when the anti-surface antiserum was employed. In all circumstances, analysis of such immunoprecipitates revealed the H2Dd spots, described above, and comparable amounts of species judged to be H2K”, since (1) they behave in a largely analogous manner and (2) they appear to comigrate with H2Kd spots studied by Jones (1977). Thus, at the end of the pulse, four major H2 spots were seen (Fig. 3(a)). Two comigrate with H2Dd, illustrated in Figure 2 (a), and the other two are considered to be H2Kd for the reasons given above. During 45 and 120 minutes of chase, these spots acquired an increasingly acidic isoelectric point and a slightly decreased sodium dodecyl sulfate/polyacrylamide gel mobility, so as to give rise to two rows of five to six spots plus a limited number of satellite spots (Fig. 3(b)). That these were indistinguishable from surface molecules was shown by analysis of antisurface. immunoprecipitates from the lysates of surface-radioiodinated cells, which showed a comparable double row of spots (Fig. 3(c)). When either 125l-labeled cells or 120-minute chased L3% Imethionine-labeled cells were treated with neuraminidase, the three to four most acidic members of each row were eliminated with a corresponding increased intensity of the original quartet (15 min pulse) and the least acidic spots apparently derived from them (Fig. 3(d) and (e)). When monensin was present during the chase (Fig. 3(f)), the pattern observed largely resembled that obtained after treatment with neuraminidase; indicating that monensin interfered with the completion of glycosylation of these molecules. The slight difference between Figure 3(f) (monensin) and Figure 3(e) (neuraminidase) shows that the block due to monensin is somewhat incomplete. FIG, 3. Two-dimensional sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by autoradiography of anti-surface immunoprecipitates. (a) CMls were pulselabeled for 15 min with [ 35S)methionine and lysed at once. In the 2-dimensional analysis of the immunopreripit,ates the 4 spots indicat,ed by arrowheads are identified as H2. By comparison with Fig. 2 and the publication of *Jones (1977) the lower 2 spots (upward pointing arrowheads) are H2Dd and the upper 2 spots (downward pointing arrowheads) are H2K”. After a 2 h chase ((b)) the indicated spots of (a) are of reduced intensity and in their place is a double family of 5 to 6 more acidic spots (small arrows), the most acidic of which are co-isoelectric with actin. (c) Surface-iodinated cells. The double row of H2 species comigrates with the double row of (b). When such iodinated cells are treated with neuraminidase ((d)), the H2 species collapse t,o regenerate the original spots (indicated in (a)) as well as lesser amount,s of more acidic neighboring spot,s. When the immunoprecipitats analyzed in (b) is treated with neuraminidase, it acquires a 2-dimensional pattern very similar to (d). This pattern is given in (e). When the chase incubation is in the presence of 1 PM-monensin, the pattern illustrated in (f) is obtained, i.e. similar to (e). but with small amounts of the H2 species more acidic than those seen in (e). These minor species can be eliminated by treatment with neuraminidase of the immunoprecipitate (not shown). The origin of the satellite spots labeled “s” is not known. They appear to comigrate with minor species described by Jones (1977) but might be due to proteolysis or correspond to H2L” (Coligan el al., 1980). “A” is actin.

PIG. 3

532

A. TARTAKOFF,

D. HOESSLI

AND

P. VASSALLI

Treatment with neuraminidase (either of intact cells or of the immunoprecipitates themselves) of the cell samples incubated with monensin during the chase eliminated these slight differences (not shown). To investigate whether monensin interfered with the transport of H2 molecules (i.e. whether the chains which remain incompletely glycosylated in the presence of monensin were able to reach the cell surface), lymphoma cells were incubated for six hours in the presence of monensin, a treatment that was found not to decrease cell viability, and then radioiodinated. The pattern of the iodinated spots was identical to t,hat of the control cells incubated without monensin. There was no preferential expression of the more basic members at the ceil surface. However, on a per cell basis, quantitation of H2 radioactivity showed that the intensity of labeling was reduced - 70”/;,, suggesting that surface H2 molecules had been lost during the six hours and not, replaced in the monensin-treated cells (not shown).

(c) Electron microscopy Treatment with monensin of either the lymphocytes or lymphoma cells has a profound effect on the Golgi complex, as has been documented for several other cell types (Tartakoff & Vassalli, 1977,1978). In the place of compressed Golgi cisternae, massively dilated smooth-surface vacuoles were observed. Like the compressed cisternae of control cells, they appear to adhere to each other along a substantial portion of their perimeter. Characteristic pictures of the lymphoma are given in Figure 4.

4. Discussion The present study investigates whether membrane Ig and H2 heavy chains pass through the Golgi subsite that is defined by the inhibitory action of monensin and seeks to gain detailed information on stages of oligosaccharide maturation of these surface glycoproteins. In principle, this approach should make it possible to ascribe specific events of oligosaccharide maturation to distinct Golgi subcompartments, since we have shown that the action of monensin is of topographic and not enzymologic origin: that is, it arrests secretory protein transport proximal to the terminal sugar transferases. It does not inhibit the transferases themselves (Tartakoff & Vassalli, 1979). In the absence of perturbant, the biosynthetic labeling experiments show that p,,, becomes retarded in its one-dimensional gel mobility during periods of time comparable to those required for transport to the cell surface. This retardation has been reported and has been ascribed to terminal sugar addition (Vassalli et al., 1980). We demonstrate here that it is blocked by monensin. For H2, the data are more extensive, as two-dimensional gel procedures were used. During chase intervals, the shift of radioactivity to generate a double family of more acidic spots is thought to result partially from sialic acid addition (since neuraminidase largely returns the chase distribution of radioactive spots toward the initial quartet) and partially to unidentified modifications (e.g. addition of galactose or fucose, phosphorylation (Rothbard et al., 1980), or attachment of sialic acid through v. chderae neuraminidase-insensitive linkages (Drzeniek, 1973)). In

MEMBRANE

GLYCOPROTEIN

TRANSPORT

533

FIN:. 4. Thin section of the lymphoma before ((a)) and after ((b)) a 1 h incubation with 1 PM-monensin. Note the selective alter&ion of the smooth-surfaced membranes of the Golgi complex (GC), which become dilated. They continue to adhere to each other. The mitochondria (M) are condensed. Other aspects of ultrastructure are unchanged. The bar represents 1 pm.

534

A. TARTAKOFF,

D. HOESSLI

AND

P. VASSALLI

the presence of monensin during chase incubation, only those shifts of radioactivity occur that cannot be ascribed to sialic acid addition. These shifts are considered to reflect covalent modifications that occur either within the rough endoplasmic reticulum or relatively proximal Golgi subcompartments. That is, although these membrane glycoproteins remain anchored to the lipid bilayer, monensin has an inhibitory effect on their terminal glycosylation, which is altogether comparable to that observed for soluble secretory proteins?. By analogy with our studies of secretory proteins (Tartakoff & Vassalli, 1977,1978) one would expect the CL,,, and H2 species bearing truncated oligosaccharides not to reach the cell surface. This point was examined by incubating cells in control medium or with monensin, iodinating them, and performing one- or two-dimensional gel analysis. As expected, only the same iodinatable H2 and TV,,,species were detected as in controls. In the case of the lymphoma cells, the cell population is nearly homogeneous and as the doubling time of the cells is - 18 hours, the six-hour period of treatment with monensin should definitely have revealed truncated species at the cell surface, had they been transported. They were not detected. It is striking that there is a progressive corresponding diminution of surface iodinatable H2 during treatment with monensin. Such a result would be anticipated if monensin stops delivery of H2 to the cell surface, but allows the turnover of previously surface-exposed molecules to proceed. A portion of this turnover has been attributed to “shedding” to the surrounding medium (Emerson et al., 1980). In the case of surface Ig transport, our observations are not conclusive, since iodination experiments address the nature of the totality of exposed p,,,, while the rapidly turning-over CL,,,followed in biosynthetic labeling experiments may derive from a minor cell population (Melchers et al., 1975). Thus, by two independent criteria (oligosaccharide maturation, arrival at the cell surface), we can conclude that two classes of surface glycoproteins (IgM, H2 heavy chains) normally pass through the Golgi subsite defined by monensin while on their way to the cell surface, and that they acquire at least the terminal sugar, sialic acid, distal to this site. Monensin

has recently

viral envelope glycoproteins

been shown

to interrupt

the intracellular

transport

of

et aE., 1980 ; Strous & Lodish, 1980) and of the acetylcholine receptor and acetylcholinesterase (Rotundo & Fambrough, 1980). Some partial characterization of the influence of monensin on oligosaccharide maturation of the viral glycoproteins has also been reported (Johnson & Schlesinger,

1980; Strous

(KliGriSinen

& Lodish,

1980).

We thank MS H. Detraz and M. Poincelet for their skilful technical assistance, Dr P. Robinson for the kind gift of the alloantiserum and Dr J. R. L. Pink for help in several pilot experiments. This work was supported by grant 3.324.78 from the Swiss National

Science Foundation.

t Comparable studies have utilized 0.1 mwcolchicine, an inhibitor of intracellular transport of many but not all secretory proteins (Ehrlich etal., 1974; LeMarchand et al., 1973; Redman etal., 1975; Tartakoff, 1980). The drug was without effect on carbohydrate maturation of pm and HZ (A. Tartakoff & D. Hoessli. unpublished observation).

MEMBRANE

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tX3.5

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Edited

by K. Simons