the of no. of 1987 by for i~.s.a. transport of the ... · cell-free transport into trans golgi...

0 1987 by The American Society for Biochemistry and Molecular Biology, Inc. T H E JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 26, Issue of September 15, pp. 12502-12510 1987

Printed in I~.s.A.

Transport of the Vesicular Stomatitis Glycoprotein to trans Golgi Membranes in a Cell-free System*

(Received for publication, January 9, 1987)

James E. Rothman From the Department of Biochemistry, Stanford University Medical Center, Stanford, California 94305

Terminal steps in the transport of the vesicular stomatitis virus glycoprotein (G protein) in the Golgi stack have been reconstituted in a cell-free system. Incor- poration of sialic acid into the oligosaccharide chains of G protein was used to monitor transport into the trans Golgi compartment. Transport-coupled sialylation required cytosol, ATP, an N-ethylmaleimide-sensitive factor extractable from Golgi membranes, and long chain acyl coenzyme A. The G protein receiving sialic acid in the cell-free system begins its in vitro transport bearing galactose residues acquired in uiuo. Earlier reports (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984a) Cell 39, 406-416) documented that transport of G protein into the medial (GlcNAc Transferase-containing) compartment is reconstituted under the same conditions. On the basis of the results reported here, it now appears that a more complete set of transport operations of the Golgi stack may be simultaneously reconstituted.

The Golgi stack consists of a series of distinct compartments (Farquhar, 1985; Dunphy and Rothman, 1985). Trans- ported proteins enter the Golgi stack from the endoplasmic reticulum at the cis compartment and move to the medial and then to the tram compartment before they diverge for separate transport to the cell surface, the lysosomal compartment, or secretory storage vesicles. The transport of proteins into and within the Golgi stack appears to be due to rounds of budding and fusion of transport vesicles (Rothman, et al., 1984a; Griffiths and Simons, 1986; Pfeffer and Rothman, 1987).

The budding of these transport vesicles has been reconstituted in a cell-free system (Fries and Rothman, 1980; Balch et al., 1984a). Incubation of isolated Golgi stacks from CHO’ cells results in the budding of a population of nonclathrin- coated vesicles from the cisternae in a reaction that requires cytosol and ATP (Balch et al., 1984b; Orci et al., 1986). When the Golgi stacks are isolated from VSV-infected CHO cells, the vesicles that bud in uitro contain the viral-encoded G protein, a glycoprotein that is transported through the Golgi in vivo (Orci et al., 1986). This suggests that the budding vesicles are indeed transport vesicles.

A complete round of transport between successive com-

* This work was supported by National Institutes of Health Grant AM 27044. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: CHO, Chinese hamster ovary; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; VSV, vesicular stomatitis virus; DTT, dithiothreitol; NEM, N-ethylmaleimide; NSF, NEM-sensitive factor; TGN, trans Golgi network.

partments of the Golgi occurs during these incubations (Balch et al., 1984a; Braell et al., 1984). The reconstituted transport corresponds to transport processes observed in uiuo (Fries and Rothman, 1980; Rothman et al., 1984a) and has been studied in some detail (Balch et al., 1984b; Wattenberg et al., 1986; Glick and Rothman, 1987; Dunphy et al., 1986; Paquet et al., 1986). To measure a complete round of transport, two distinct types of Golgi membranes are incubated together with cytosol and ATP (Balch et al., 1984a; Balch and Rothman, 1985). One population of Golgi stacks is from VSV-infected CHO clone 15B cells, termed the “donor.” The 15B mutant of CHO cells is missing the Golgi enzyme GlcNAc transferase I (Tabas and Kornfeld, 1978) and is thus unable to incorporate GlcNAc into cellular glycoproteins or into G protein in VSV-infected cells. Thus, donor Golgi contain G protein but cannot add GlcNAc to its oligosaccharide chains. The second population of Golgi is from uninfected wild-type CHO cells, termed the “acceptor.” When donor and acceptor stacks are incubated together in the presence of cytosol and ATP, the G protein- containing vesicles that bud from the donor Golgi stacks (Orci et al., 1986) can potentially transport G protein to the GlcNAc transferase-containing acceptor stacks, resulting in the transport-coupled incorporation of GlcNAc into G protein. UDP- [3H]GlcNAc is added to the incubation, and transport is monitored by counting the G protein isolated by immunoprecipitation after the assay is completed (Balch et al., 1984a; Balch and Rothman, 1985).

Because the enzyme GlcNAc transferase I is located in the medial Golgi compartment (Dunphy et al., 1985), the assay just described exclusively measures transfers into the medial compartment of the acceptor Golgi, presumably via vesicles budding from the cis cisternae. Yet, a careful analysis of serial sections of incubated Golgi stacks revealed that the G protein- containing vesicles bud from every cisterna in each stack, independent of the level (Orci et al., 1986). This would suggest that our incubation conditions may reconstitute transport at multiple levels of the Golgi stack simultaneously.

To test this I have altered the means of monitoring transfer, although not the basic incubation conditions used, with the intent of measuring possible transfers into the tram Golgi compartments of the acceptor Golgi. The idea is simply to monitor the incorporation of r3H]sialic acid (rather than [3HJ GlcNAc) into G protein during the incubations, taking advan- tage of the fact that sialic acid is added in the tram Golgi compartment (Roth et al., 1985). To accomplish this, it is necessary to use as a donor of G protein Golgi membranes incapable of incorporating sialic acid to assure that all sialylation is due to transport to the acceptor Golgi. For this purpose I have employed the Golgi fraction from VSV-infected CHO clone 1021 cells (Briles et al., 1977) as the donor and initially the Golgi fraction of wild-type CHO cells as acceptor. Clone 1021 cells are missing a permease that normally allows CMP-sialic acid to enter the Golgi lumen

12502

Cell-free Transport into trans Golgi 12503

(Deutscher et al., 1984). Thus, even though the 1021 cells have a normal battery of sialyltransferases (Briles et al., 1977), they are unable to incorporate sialic acid into glycoproteins and glycolipids.

Extensive studies from my laboratory have established by subcellular fractionation (Dunphy et al., 1981) and by using cell fusion techniques (Rothman et al., 1984a, 1984b) that GlcNAc is added in a separate and earlier "medial" Golgi compartment than galactose and sialic acid ("trans") in CHO cells. Indeed physical movement by vesicles is required for protein movement between these two glycosylation sites. This was demonstrated by the "hopping" of the VSV G protein from the site of GlcNAc incorporation in one Golgi stack to the sites of galactose and sialic acid incorporation in another Golgi stack after cell fusion (Rothman et aL, 1984b). Such movements were found to be vectorial.

EXPERIMENTAL PROCEDURES

Materiuk Unless described otherwise, all chemicals were obtained as de-

scribed previously (Balch et a i , 1984a). UDP-I3H]GlcNAc (25 Ci/ mmol), UDP-(3H]galactose (50 Ci/mmol), and CMP-[3H]sialic acid (19 Ci/mmol) were from Du Pont-New England Nuclear. Slug lectin (Lirnax fluuus agglutinin) was the gift of Dr. Ronald Miller, Medical University of South Carolina, Charleston, SC.

Methods General-CHO cells, virus, and antiserum to G protein were grown

or derived as described previously (Balch et al., 1984a; Balch and Rothman, 1985). CHO clones 1021,13, and 15B were kindly provided by Prof. Stuart Kornfeld of Washington University (St. Louis, MO). The 1021 cells were infected with VSV for preparing donor membranes exactly as described for VSV-infected 15B cells (Balch et al., 1984a; Balch and Rothman 1985). Infected and uninfected CHO cells were homogenized and the Golgi fractions (typically 0.5-0.7 mg/ml protein) prepared using sucrose gradients exactly as described (Balch et al., 1984a). CHO cytosol (typically 5-7 mg/ml protein) was prepared and gel-filtered on Sephadex G-25 as described (Balch et al., 1984a). All fractions were frozen in liquid N2 and stored at -80 "C. Protein was measured according to Lowry et al. (1951).

Standard Assay for Transport-coupbd Sialylation of G Protein- Reactions (50-pl final volume) contained 25 mM HEPES (pH 7.0 with KOH), 15 mM KCl, 2.5 mM magnesium acetate, 5 mM D m , 50 pM ATP, 250 phf UTP, 2 mM creatine phosphate, 7.3 IU/ml rabbit muscle creatine phosphokinase, 10 pCi/ml CMP-[3H]sialic acid, 5 p1 of donor Golgi fraction (from VSV-infected 1021 cells in 1 M sucrose, 10 mM Tris-HC1, pH 7.41, 5 p1 of acceptor Golgi fraction (from uninfected 15B CHO cells in the same 1 M sucrose buffer), and 5 p1 of 15B cell cytosol fraction. After 2 h at 37 'C, 50 pl of a "detergent buffer" (50 mM Tris-HC1, pH 7.5, 250 m M NaCl, 1 mM Na,EDTA, 1% Triton X-100, and 1% sodium cholate) was added along with 10 p l of rabbit anti-G antiserum. After at least 4 h at 4 "C, the immunoprecipitates were collected on Millipore filters and counted as described (Balch et al., 1984a).

RESULTS

Acquisition of Sialic Acid by VSV G Protein during Cell-free Incubations of Golgi Fractions-To test for the transfer of VSV G protein from donor Golgi membranes to the sialic acid transferase-containing trans cisternae of acceptor Golgi stacks, the Golgi fraction from VSV-infected CHO clone 1021 cells (donor) was incubated with the Golgi fraction from uninfected wild-type CHO cells (acceptor), as well as a CHO cytosol fraction, ATP and an ATP-regenerating system, and CMP-[3H]sialic acid (2 pCi/ml) in the same buffer previously used (Balch et al., 1984a) to reconstitute transport to the medial Golgi compartment (25 mM HEPES, pH 7.0, 25 mM KCl, 2.5 mM magnesium acetate). After an incubation of 90 min at 37 "C, detergent was added along with an antibody to G protein, and the quantity of [3H]sialic acid incorporated

into the oligosaccharide chains of G protein was determined by counting the immunoprecipitate that had been collected on a Miliport filter (Table I).

A total of 176 cpm was incorporated in this complete incubation. Omission of either the donor, the acceptor, cytosol, or ATP greatly reduced glycosylation. The sialylated G protein was retained in sealed Golgi vesicles such that the immunoprecipitable radioactivity was resistant to external tryptic attack unless the vesicles were disrupted with Triton (Table I). Sodium dodecyl sulfate gel analysis (data not shown) revealed that the external tryptic attack cleaved off the normally cytoplasmic carboxyl-terminal domain of all of the sialylated G protein, as would be expected (Balch et al., 1984a). All of these properties are characteristic of the transport of G protein from cis to medial Golgi that we have previously found to be reconstituted in the same kind of incubations (Balch et al., 1984a). Therefore, it is reasonable to suggest that the addition of sialic acid to G protein reflects its transfer from the 1021 donor Golgi (unable to incorporate sialic acid) to the trans compartment in the acceptor Golgi. Transport-coupled sialylation was also temperature-dependent, not occurring at ice temperature.

The 3H in the immunoprecipitate accurately reflects incorporation into G protein (Table I) since the amount of 3H retained by the filter is greatly reduced when a nonimmune serum is used in place of an anti-G serum or when no G protein is present in the incubation (such as when the donor fraction is omitted).

To attempt to improve the extent of sialic acid incorpora-

TABLE I Specificity of transport-coupled sialybtwn of G protein

The complete incubation contained in a 50-pl volume, 5 pl of donor (VSV-infected 1021 Golgi fraction), 5 pl of acceptor (uninfected wild- type Golgi fraction), 5 p l of cytosol from wild-type CHO cells, 50 p~ ATP, 250 p~ UTP, 2 mM creatine phosphate, 7.3 IU/ml creatine kinase in 25 mM HEPES (pH 7), 15 mM KCl, 2.5 mM magnesium acetate, 2 pCi/ml CMP-[3H]sialic acid, without DTT. After 90 min at 37 "C, assays were stopped and immunoprecipitated with anti-G serum. Immunoprecipitates were counted for 5 min each. 1) Complete incubation as just described. 2) Omission of only ATP, UTP, creatine phosphate, and creatine kinase. 3) Omission of cytosol. 4) Replace- ment of acceptor fraction with 5 pl of 1 M sucrose. 5 p l of donor was added after the incubation was terminated to provide a pool of unlabeled G protein for immunoprecipitation. 6) Incubation at 0 "C rather than 37 "C for 90 min. 7) Nonimmune serum replacing anti-G serum. 8) After a complete incubation, 2 mM NalEDTA and 100 pg / ml trypsin were added. After 10 min at 37 "C, 200 pg/ml soybean trypsin inhibitor was added before immunoprecipitation. 9) Same as 8 except that trypsin inhibitor was mixed with trypsin and added together with the EDTA. 10) Same as 8 except that 0.1 % Triton X- 100 was added together with trypsin.

Immuno- Condition precipi-

tate

cpm 1. Complete incubation 176 2. Complete incubation minus ATP, UTP, and 13

creatine phosphate 3. Complete incubation minus cytosol fraction 6 4. Complete incubation minus wild-type Golgi 7

5. Complete incubation minus VSV-infected 1021 7

6. Incubation on ice 0 7. With preimmune serum replacing a n t i 4 14

protein serum in immunoprecipitation 8. Trypsin added after incubation 184 9. Trypsin inhibitor added before trypsin after 185

10. Trypsin plus Triton X-100 after incubation 7

acceptor

Golgi donor

incubation

12504 Cell-free Transport into trans Golgi

tion, the conditions for cytosol and ATP-dependent sialylation were optimized. Transport was stimulated about $fold by adding dithiothreitol (Fig. 1) in contrast to the lack of effect of DTT on transport to medial Golgi (Balch and Roth- man, 1985). The buffer conditions chosen based on the salt and magnesium dependence (Figs. 2 and 3) consisted of 15 mM KCI, 2.5 mM magnesium acetate, 25 mM HEPES-KOH (pH 7.0), and 5 mM DTT. These conditions are very similar to those found optimal for transport to medial Golgi (Balch and Rothman, 1985), with the exception of the presence of DTT and the slightly lower level of KC1 (15 mM uersuS 25 mM for medial transport). In addition, the amount of [3H] sialic acid incorporated was found to be proportional to the amount of CMP-[3H]sialic acid added up to about 1 p~ (equivalent to about 20 pCi/ml) (data not shown). This is the case because the pool of endogenous unlabeled CMP-sialic acid is between 1 and 2 p ~ , as revealed by isotope competition (Fig. 4). A concentration of CMP-[3H]sialic acid of 0.5 p~ (10 pCi/ml) is added in routine incubations.

0 t

;b Goo

0:: ;z

4

-It

- "? I

0 1 1 1 1 1 1 1 1 I

0 2 4 6 8 10 IDTTI (mM)

FIG. 1. Incorporation of [3H]sialic acid into G protein as a function of the concentration of DTT, at 3 mM magnesium acetate, and 4 rCi/ml CMP-[3H]sialic acid in otherwise standard assays. Arrow indicates the value chosen as optimal.

0 5 10 [MgAcl (mMJ

FIG. 2. Incorporation of [3H]sialic acid into G protein as a function of the concentration of magnesium acetate in the presence (0) or the absence (0) of cytosol at 4 pCi/ml CMP- [3H]sialic acid in otherwise standard assays. Arrow indicates the value chosen for standard assays as optimal to minimize the nonspecific (i.e. cytosol-independent) incorporation that begins to occur at higher concentrations of magnesium.

I I I I 1 1

0 20 40 60 80 100 [KCII (mM)

FIG. 3. Incorporation of [3H]sialic acid into G protein as a function of the concentration of KC1 in otherwise standard assays. Arrow indicates the value chosen as optimal.

" 0 2 4 6 8 1 0 1 2

UNLABELLEO CMP-SIALIC ACID ADDED (CIM)

FIG. 4. Isotope competition experiment. Inhibition of incorporation of (3H]siaIic acid by unlabeled CMP-sialic acid. All assays contained the standard 10 pCi/ml (0.2 ptM) CMP-[3H]sialic acid plus the indicated concentration of unlabeled CMP-sialic acid.

A further increase in the efficiency of sialylation came from the use of the Golgi fraction from uninfected 15B CHO cells as acceptor in place of the Golgi fraction from wild-type cells. The Golgi fraction from 15B cells should have the capacity to incorporate sialic acid (Tabas and Kornfeld, 1978). Therefore the 15B Golgi should, in principle, be able to replace the wild- type Golgi as acceptor for transport-coupled sialylation. Fig. 9 shows that this was indeed the case and surprisingly that the 15B Golgi are nearly twice as efficient as acceptors as the Golgi from wild-type cells. Although there is no obvious explanation for this, because of this finding the 15B Golgi are used routinely as acceptor in the standard transport-coupled sialylation assay. With all of the above improvements, the standard assay routinely results in the incorporation of between 500 and 1000 cpm, depending on the membrane preparation. Monensin (10 p ~ ) was without effect (data not shown).

Fig. 5 shows that the efficiency of transport increases linearly with the concentration of cytosol added until a pla- teau value is reached. Bovine brain and CHO cytosol act almost equivalently in promoting transport. Fig. 8 shows that


300 L

0 CYTOSOL FROM BOVINE BRAIN

n 1 I I I I 1-

0 20 40 60 80 100 CYTOSOL ADDED Ipo)

FIG. 5. Cytosol dependence. Incubations (50 pi) were for 90 min at 37 "C in otherwise standard assays using VSV-infected 1021 Golgi as donor and 15B Golgi as acceptor. Open circles, cytosol from bovine brain prepared as described (Wattenberg and Rothman, 1986). Closed circles, cytosol from 15B CHO cells. Samples were counted for 10 min each. A background of 94 cpm (for an incubation without cytosol) has been subtracted.

1 2 3 4 5 TIME (hours)

FIG. 6. Time course. Samples (50 ~ 1 ) of a standard incubation were stopped at the time indicated, and the amount of [3H]sialic acid incorporated into G protein was determined after immunoprecipitation.

the extent of transport is proportional to the concentration of VSV-1021 donor. Fig. 9B shows that the transport is a saturable function of the concentration of 15B acceptor and that the Golgi fraction from uninfected 1021 cells cannot act as acceptor for transport-coupled sialylation. This implies that the in vivo defect in 1021 cells (lack of Golgi permease for CMP-sialic acid; Deutscher et al., 1984) is retained in vitro under the assay conditions. Note that while 1021 Golgi do not act as an apparent. acceptor for transport-coupled sialylation, they do act (Fig. 9A) as acceptor for transport-coupled N - acetylglucosaminidation (medial transport assay).

Fig. 6 shows the time course for the transport-coupled sialylation. After a lag of about 15 min, sialylation proceeds linearly for about 2 h, after which it slowly begins to cease. The lag phase of 15 min reflects the time taken for transport

intermediates to reach steady-state levels, rather than a lag in sialylation. This was clear from a preincubation experiment (Fig. 7 ) in which donor, acceptor, cytosol, and ATP were preincubated for 20 min without CMP-[3H]sialic acid to allow the transport intermediates to form before the sugar nucleotide was added. Sialylation now began virtually without a lag after adding CMP-[3H]~ialic acid (Fig. 7 ) . Had the lag been due to the time required for uptake of the sugar nucleotide into the Golgi lumen or for a gradual sialylation, then the lag would still have been present in spite of the preincubation. A similar lag, also due to the time taken for the production of transport intermediates, has been reported for the medial Golgi reconstitution (Balch et at., 1984a) and dissected into several stages in some detail (Balch et al. 1984b; Wattenberg et al., 1986).

The G Protein Receiving Sialic Acid in Vitro Consists of a Transiting Pool en Route to the Plasma Membrane in Vivo- The donor fraction, housing the G protein that receives sialic acid upon incubation with acceptor Golgi in vitro, consists of a Golgi-enriched fraction whose principal contaminants are likely to be plasma membranes and endosomes. Therefore, it is possible that the G protein sialylated in the tram compartment of the acceptor Golgi derives from endosomes or even plasma membrane contaminants of the Golgi donor fraction and not from the Golgi cisternae in this fraction. To test for this, the donor Golgi fraction was prepared from VSV-infected CHO 1021 cells after a 30-min incubation with cycloheximide to inhibit G protein synthesis. This incubation will largely deplete G protein from the Golgi stack (since G is transported onto the surface but is no longer received by the Golgi) (Rothman et al., 1984b; Balch et al., 1984a). However, the pool of G protein at the cell surface (and the pool in the endocytic pathway that derives from the cell surface pool) will not be significantly reduced by this brief treatment.

Fig. 8 shows that the donor activity of the Golgi fraction is lost after protein synthesis is blocked. This implies that G protein receiving sialic acid originates in a transiting intra- cellular pool en route to the plasma membrane and not from plasma membrane or endosomal contaminants in the donor Golgi fraction. In all likelihood, the pool of donated G protein

m . l , I I I I I 1

300- 20 rnin without

Pnincubmd

200 -

4 5 10 15 20 25 30 35 40 I TIME AFTER ADDING

SIALIC ACID CMP-h- CMP-3H-SIALIC ACID lrnin)

FIG. 7. Effect of preincubation without CMP-[SH]sialic acid upon subsequent time course. Triangles, a standard incubation in which CMP-[3H]sialic acid (10 pCi/ml) was present from time zero. Samples of 50 ~1 were immunoprecipitated. Circles, an otherwise standard incubation was composed without CMP-[3H]sialic acid. After a preincubation of 20 min at 37 "C, 10 pCi/ml CMP-[3H]sialic acid were added from a 200 pCi/ml stock solution. Samples of 50 pl were taken for immunoprecipitation at the times thereafter indicated on the x axis.


0 1 1 0 0.6 1.0 1.5 2.0 2.5 3.0

VSV/1021 DONOR MEMBRANE ADDED (pa)

FIG. 8. Blocking protein synthesis depletes G protein from the donor compartments. Closed circles, titration of a standard donor preparation from VSV-infected 1021 cells. Shown is the amount of [3H]sialic acid incorporated into G protein in otherwise standard 50-pl incubations as a function of the quantity of donor Golgi fraction added (in pg of protein). The final concentration of sucrose was maintained at 0.2 M in all incubations. Open circles, cycloheximide (100 pg/ml) was added to the VSV-infected 1021 cells for 30 min at 37 "C before homogenization and preparation of the donor Golgi fraction. This experiment was done in parallel with the control donor preparation (clased circles). To assure identical conditions in the immunoprecipitation of G protein from each assay, an appropriate amount of the control donor and cycloheximide-donor preparations was added after the incubation such that a total of 2.6 pg of each were present at the time of immunoprecipitation.

originates in the Golgi membranes themselves. In support of this, the extent of G protein transport to trans

Golgi can be roughly calculated from the amount of sialic acid incorporated. Assume an average of two sialic acids is incorporated into each G protein receiving sialic acid (an average of one at each of the two asparagine-linked oligosaccharide chains, as in CHO cells in vivo (Rothman et al., 1984a) and an endogenous pool size of 2 p~ CMP-sialic acid (estimated from the concentration of unlabeled CMP-sialic acid that eliminates half of the incorporation by isotope competition in Fig. 4). About 1000 cpm of [3H]sialic acid are incorporated into G protein in a 2-h incubation (with a scintillation counting efficiency of about 25% on filters). This is clearly an underestimate of the full extent of sialylation (Fig. 6) and corresponds to about 0.2 pmol of G protein transported from the donor Golgi fraction (about 3 pg of protein routinely). For comparison, we previously estimated that about 0.2 pmol of G protein is transported from the cis to the medial Golgi compartment from 2.5 pg of donor Golgi membrane and that this quantity of G protein accounts for about 25% of the total G protein in the Golgi fraction (Balch and Rothman, 1985). Since almost all of the Golgi membranes in our preparations are in the form of stacks of cisternae (Braell et al., 1984), it follows that the G protein that receives sialic acid in vitro originates in stacks of cisternae in the donor fraction. Evi- dently, transport to tram Golgi in vitro is at least as efficient as transport to medial Golgi in vitro. However, the higher pool of endogenous CMP-sialic acid results in a relatively low incorporation of [3H]sialic acid as compared to [3H]GlcNAc.

The G Protein Receiving Sialic Acid in Vitro Has Already Received Galactose in Vivo-At what level in the donor Golgi stack does the G protein receiving sialic acid during transport in vitro originate? To help find out, we can ask whether or not the sialylated G protein has also incorporated GlcNAc (a

marker of the medial compartment) or galactose (a marker of the trans compartment) during its transport. For this purpose, the sugar nucleotide precursors UDP-[3H]GlcNAc, UDP-[3H] galactose, and CMP-[3H]sialic acid were added at 20 &i/ml (10 times the concentration of UDP-[3H]GlcNAc used to detect GlcNAc incorporation in routine medial Golgi assays) to parallel incubations of donor (from VSV-infected 1021) with or without acceptor (from uninfected 15B). G protein was then isolated by immunoprecipitation, digested exhaus- tively with Pronase, and the glycopeptides precipitated with the sialic acid-specific (Miller et al., 1982) slug lectin (Table 11). Although [3H]sialic acid was easily detected in the sialylated glycopeptides derived from G protein, there was no significant acceptor-dependent incorporation of either [3H] GlcNAc or [3H]galactose. Fig. 10 will show that [3H]galactose and [3H]GlcNAc are even more efficiently incorporated than [3H]sialic acid into bulk endogenous glycoprotein acceptors in the Golgi membranes under these incubation conditions. Therefore, the inability to detect incorporation of either GlcNAc or galactose into the G protein sialylated in vitro strongly suggests that the sialylated G protein already pos- sessed both its GlcNAc and galactose residues at the start of the incubation.

TABLE I1 Lack of incorporation of GlcNAc and galactose into G protein

sinlylated during cell-free incubations Each incubation (100-pl final volume) contained 10 11 of donor

(Golgi fraction from VSV-infected 1021 cells) and 20 pl of wild-type CHO cytosol in the standard transport-coupled sialylation assay buffer with ATP, UTP, and the ATP-regeneration system, either with 10 p1 of acceptor (Golgi fraction from uninfected CHO 15B cells, indicated by a "plus") or without acceptor fraction (indicated by a "minus," in which case 10 pl of 1 M sucrose was added instead). A final concentration of 20 pCi/ml sugar nucleotide was also added, either UDP-['IGlcNAc (for 1 and 2), UDP-[3H]galactose (for 3 and 4), or CMP-[3H]sialic acid (for 5). After 90 min at 37 "C, incubations were stopped with 100 pl of "detergent buffer" (see "Experimental Procedures") and 20 pl of anti-G serum. After 16 h at 4 "C, 50 pl of Staphylococcus A cells (Behring Diagnostics, washed three times in "detergent buffer" and resuspended in their original volume) were added. After 1 h at 4 "C the Staphylococcus A cells were pelleted and washed three times in detergent buffer, suspended in 200 pl of 1% sodium dodecyl sulfate, 15 mM DTT, 50 mM Tris-HC1 (pH 6.8), and boiled. Triton X-100 (20 pl of a 2% solution) was added to the supernatant as carrier, and protein was precipitated with 200 p1 of ice-cold 20% trichloroacetic acid. The precipitate was pelleted, washed with ice-cold acetone, and air dried. Then, 50 pl of Pronase (20 mg/ml in 0.1 M Tris-HC1 (pH 7.5) with 10 mM CaCL) were added. After 1 day at 50 "C to digest the immunoprecipitated G protein, the digest was boiled and the supernatant (containing G protein-derived glycopeptides) was saved. A 10-pl sample of each glycopeptide preparation was incubated with 20 pl of 4 mg/ml slug lectin (L. flaous agglutinin, dissolved in 0.1 M NaCl, 50 mM Tris-HC1, pH 7.5) for 15 min at 20 "C. Then 1 ml of ice-cold 4 M (NH&SO, containing 0.01% bovine serum albumin and 50 mM Tris-HC1 (adjusted to a final pH of 7.0) was added to precipitate the sialic acid-containing glycopeptides that had bound to the lectin. The precipitate was collected by centrifugation, dissolved in 1 ml H20, and counted for 5 min. Shown is the amount of [3H]GlcNAc (1 and 2), [3H]galactose (3 and 4), or [3H]sialic acid (5) that had been incorporated into oligosaccharides of G protein that also received sialic acid in vitro.

'H sugar recovered

Incubation containing Acceptor in sialic

glycopeptides from acid-containing donor and

G protein

cpm 1. UDP-[3H]Gl~NA~ - 31 2. UDP-['H]G~CNAC + 48 3. UDP-['H]galactose - 20 4. UDP-[3H]galactose + 36 5. CMP4'Hlsialic acid + 188


To confirm in an independent fashion that the G protein transferred from the donor Golgi membrane to the acceptor Golgi for sialylation already has its galactose at the start of the incubation, two further tests were applied. In the first test, the effects of adding UDP-galactose and depleting endogenous pools of UDP-galactose were determined (Table 111). Adding UDP-galactose (up to 0.6 mM) was without stimulatory effect upon transport-coupled sialylation of G protein. Also, an enzymatic depletion of 90% of the endogenous pool of UDP-galactose did not diminish transport-coupled sialylation (Table 111). This depletion was accomplished by adding the enzyme UDP-glucose dehydrogenase and NAD+. The dehydrogenase catalyzes the NAD+-dependent conversion of UDP-glucose to UDP-glucuronic acid (Wilson, 1965). The endogenous UDP-galactose is depleted together with UDP-glucose because these two sugar nucleotides are in a rapid equilibrium catalyzed by UDP-galactose epimerase (present in the cytosol fraction used in the transport assays). The depletion of UDP-galactose was monitored from the conversion of added tracer UDP-[3H]galactose to UDP-[3H] glucuronic acid in parallel incubations by using thin layer chromatography (Table 111). The small (about 25%) inhibition

TABLE I11 Lack of effect of UDP-galactose on the transport-coupled sinlylatwn of

G protein To measure transport-coupled sialylation, incubations were com-

posed in the standard fashion (see "Experimental Procedures") except twice the standard concentration of cytosol (10 pl of cytosol/50-pl final volume) was used to increase the concentration of endogenous UDP-galactose epimerase. In addition, unlabeled UDP-galactose was added to incubations 2 and 3 at 100 and 600 phi, respectively; NAD+ (2 mM) and magnesium acetate (2 mM) were added to incubation 4; and 40 pg/ml UDP-glucose dehydrogenase (Sigma, Type VI from bovine liver, 0.5 unit/mg of protein) as well as NAD and magnesium acetate were added to incubation 5. After 2 h at 37 "C, the amount of [3H]sialic acid incorporated into G protein was determined by immunoprecipitation. To determine how much UDP-galactose remains after these incubation conditions, a parallel set of five incubation mixtures (10-pl final volume) was composed identically to those just described, except CMP-[3H]sialic acid was omitted, and a trace amount (1 pCi/ml) of UDP-['H]galactose was added. After the 2-h incubation at 37 "C, samples (1 pl) were removed and spotted on strips of polyethyleneimine cellulose that had been prespotted with 1 pl of a mixture of sugar nucleotide carriers (6.7 mM each of UDP- galactose, UDP-glucose, and UDP-glucuronic acid). The samples were chromatographed in 0.25 M LiCl to separate the UDP-[3H]glucuronic acid produced ( R F about 0.1) from UDP-[3H]galactose or UDP-[3H] glucose (migrating together a t RF about 0.7). The spots were located with short wave UV light, cut out, and counted. The data shown is the amount of 3H derived from UDP-[3H]galactose remaining in the combined UDP-galactose plus UDP-glucose spots expressed as a fraction of the total 3H recovered from this and the UDP-glucuronic acid spot. Note that the conversion of UDP-[3H]galactose to UDP- [3H]glucuronic acid (incubation 5) is in fact half complete in 2 min at 37 "C and complete within 10 min ( d a t a not shown).

Fraction of initial

Standard incubation, plus [ ikorported remaining

incubation

'H Sialic acid UDP-galactose

into G protein after

cpm 1. No addition 2036 0.84 2. UDP-galactose (100 pM) 1776 0.94 3. UDP-galactose (600 pM) 1580 0.95 4. NAD+ (2 mM) and magnesium 1520 ND"

5. UDP-glucose dehydrogenase 1503 0.096 acetate (2 mM)

(40 pg/ml) and NAD+ (2 mM) and magnesium acetate (2 mM) a Not determined.

observed (Table 111, line 5) is due to the effects of NAD' and not depletion via the dehydrogenase (Table 111, line 4).

The finding that UDP-galactose is apparently not required for the transport-coupled sialylation confirms the conclusion that the sialylated G protein already has galactose at the start of the in uitp6 incubation. A second independent test of this is shown in Fig. 9. The Golgi fractions from a series of CHO cell glycosylation mutants were tested as acceptors in the transport-coupled sialylation (trans compartment) and transport-coupled N-acetylglucosaminidation (medial compartment) assays. Most significmtiy, the Golgi fraction from clone 13 CHO cells is equivalent to wild-type CHO cell Golgi in acting as acceptor for transport-coupled sialylation (Fig. 9B). Clone 13 cells do not incorporate galactose into glycoproteins or glycolipids (Briles et al., 1977), most probably because these cells lack the permease for uptake of UDP-galactose into the Golgi lumen (clone 13 cells have essentially normal levels of both UDP-galactose and galactosyltransferases). Despite their inability to incorporate galactose, clone 13 Golgi none- theless sialylate transported G protein as efficiently as wild- type Golgi (Fig. 9B). This implies, independently of the other two tests, that the sialylated G protein possesses its full complement of galactose at the time the donor Golgi membranes were isolated. Fig. 10 confirms that the in oiuo defect of clone 13 Golgi is retained in uitro. Whereas clone 13 Golgi incorporate about the same levels of [3H]GlcNAc from UDP- [3H]Gl~NA~ into endogenous acceptors as do wild-type and clone 1021 Golgi (Fig. lOA), clone 13 Golgi incorporate much less [3H]galactose from UDP-[3H]galactose than either wild- type or clone 1021 Golgi membranes (Fig. 1OB). As expected, clone 1021 Golgi incorporate much less [3H]sialic acid from CMP-[3H]sialic acid than do wild-type Golgi (Fig. 1OC). The inability of clone 13 Golgi to incorporate sialic acid (Fig. 1OC) is presumably secondary to their inability to incorporate galactose, resulting in the absence of a pool of endogenous acceptors having galactose termini.

Altogether, there are three lines of evidence to suggest that the G protein sialylated by acceptor Golgi in uitro originates in the Golgi membranes within the donor Golgi-enriched fraction. First, the donor activity is found in the Golgi fraction. Second, and stronger, the G protein sialylated in uitro starts out already having galactose, implying a location within the Golgi or a post-Golgi location. Third, treatment with cycloheximide in vivo results in the rapid depletion of G protein from the donor compartment ruling out a post-Golgi location.

Transport-coupled Sialylation Requires NSF and Acyl Coen- zyme A-Treatment of donor and acceptor membranes with 1 mM N-ethylmaleimide (NEM) at 0 "C for 15 min eliminates their activity in the transport-coupled N-acetylglucosaminidation (medial) assay (Glick and Rothman, 1987). Transport is restored when a salt and ATP extract of untreated Golgi membranes is added. The restorative factor is also sensitive to NEM and has been termed "NSF" for NEM-Sensitive - Factor. Presumably, the NEM treatment of membranes quenches their endogenous supply of NSF. The ability of NSF to restore transport-coupled N-acetylglucosaminidation is greatly stimulated by the inclusion of long chain acyl coenzyme A (Glick and Rothman, 1987). To see whether a similar behavior is observed in the transport-coupled sialylation assay, the donor (VSV/1021) and acceptor (uninfected 15B) membranes were pretreated with 1 mM NEM, and then the NEM was quenched with DTT. The ability of an NSF fraction active in the medial assay (Fig. 1l.4) to restore transport in the trans assay was measured in the presence or absence of palmitoyl-CoA (Fig. 11B). Clearly, NSF and acyl coenzyme A


VSV/lSB-rcaptor amptor:

- 0 250 500 750 lo00

v s v / 1 0 2 1 - ~ 0 r

n CMP-'H-SIALIC ACID

AMOUNT OF ACCEPTOR GOLGI FRACTION (units of Gal trsnrferar d d d )

FIG. 9. Titration of Golgi fractions of wild-type and mutant CHO cells as acceptore in the transport- coupled N-acetylglucosaminidation (panel A) and sialylation (panel B) assays. Golgi fractions were prepared from uninfected wild-type and clones 15B, 13, and 1021 cells as described for wild-type CHO cells (Balch et al., 1984a). To standardize the different Golgi fractions, their concentrations of galactosyltransferase were measured. Galactosyltransferase assays (Paquet and Moscarello, 1984) contained 50 mM Tris-HC1 (pH 7.5), 20 mM MnC12, 0.5% Triton X-100, 0.5 mM UDP-galactose (5 mCi/mmol), and samples of the Golgi fraction to be assayed, with or without 20 mM GlcNAc as glycosyl acceptor, in a final volume of 50 pl. After 30 min at 37 "C, 1 ml of 1 mM NaZEDTA was added, and the mixture was filtered through a column containing 1 ml of Dowex AG 1- X8. The flow-through and wash (consisting of 1 ml of water) were counted to measure the amount of 13H] galactosyl-GlcNAc synthesized. Galactosyltransferase activity is the increment of 3H in this fraction due to the addition of GlcNAc. The specific activities of the Golgi membrane fractions used were: uninfected 15B, 320 cpm galactose transferred/pg; uninfected 13,480 cpmlpg; uninfected 1021,260 cpmlpg; uninfected wild-type, 450 cpm/ pg; VSV-infected 15B, 515 cpm/pg; VSV-infected 1021, 194 cpm/pg. Panel A, incorporation of 13H]GlcNAc into G protein in incubations of VSV-15B Golgi as donor with the various uninfected Golgi fractions as potential acceptors as a function of the amount of Golgi added to each assay (50 pl) as measured by units of galactosyltransferase (1 unit is 1 cpm of [3H]galactose transferred). The concentration of sucrose was maintained at 0.2 M independent of the amount of acceptor added. 0, clone 13 as acceptor. A, clone 1021 as acceptor. 0, clone 15B as acceptor. W, wild-type as acceptor. Assays were otherwise standard as described previously (Balch et al., 1984a) containing 2 NCi/ml UDP-[3H]GlcNAc. Each incubation contained 1420 galactosyltransferase units of VSV-15B Golgi fraction as donor. Panel B, incorporation of [3H]sialic acid into G protein in incubations of VSV-1021 Golgi as donor with the various uninfected Golgi fractions as potential acceptors as a function of the amount of acceptor added. Symbols as in p a n e l A. Each 50-pl incubation contained 630 galactosyltransferase units of VSV-1021 Golgi fraction as donor.

2.0 p7"l UDP-3H-GlcNAc

" 0 1250 2500 AMOUNT OF ACCEPTOR GOLGI FRACTION (units of 0.1 tranf0r.r)

FIG. 10. Incorporation of ['H]GlcNAc, [sH]galactose, and [%]sialic acid (SA) from sugar nucleotides into endogenous acceptors in the Golgi fractions from uninfected wild-type (m, clone 13 (0), and clone 1021 (A) CHO cells used as in vitro transport acceptors in Fig. 9. Shown is the amount of 3H sugar incorporated into acid-precipitable material as a function of the amount of Golgi fraction added (as measured in units of galactosyltransferase, see Fig. 9) in 50-pl incubations for 1 h at 37 'C. Incubations were composed as for standard transport-coupled sialylation assays (containing ATP, UTP, and ATP-regenerating system and 15B cytosol) with the 3H sugar nucleotide indicated and contained the indicated quantity of the Golgi fraction. The final concentration of sucrose was 0.2 M in all cases. Panel A, incubations contained 5 pCi/ml UDP-[3H]GlcNAc. Panel B , incubations contained 10 pCi/ml UDP-[3H]galactose. Panel C, incubations contained 10 pCi/ml CMP- 13H]sialic acid. After the incubation, 25 pl of ovalbumin (50 mg/ml) and 2 ml of 5% trichloroacetic acid were added. The precipitate was collected on glass-fiber filters, and the filters were dried and counted.

Cell-free Transport into trans Golgi

VSV/lO21+158 UDP-[3HlGlcNAc

1000 CMP-13HlSlALlC ACID Control -

12509

I I I

2 4 ”

0

NSF AODED (PI)

2 4 6

FIG. 11. Restoration of transport by NSF to NEM-treated donor and acceptor membranes‘in the transport-coupled N-acetylglucosaminidation (A) and the transport-coupled sialylation (B) assays in the presence (A) and the absence (0) of 10 p~ palmitoyl coenzyme A. Membranes were incubated with 1 mM NEM for 15 min at 0 “C in 1 M sucrose, 10 mM Tris-HCl, pH 7.4. Then 2 mM DTT was added. Then the membranes (7 pg total of donor and acceptor in all cases) were added to transport assays (50-pl final volume) containing standard concentrations of cytosol (from 15B cells, preincubated at 37 “C for 30 min to completely eliminate the endogenous cytosolic pool of NSF), ATP, UTP, and the ATP-regenerating system, and the appropriate buffers with or without palmitoyl-CoA (10 p ~ ) . The concentration of UDP-[3H]GlcNAc was 2 pCi/ml; that of CMP-[3H]sialic acid was 20 pCi/ml. The NSF (2.8 mg/ml) was prepared by extraction of a rat liver Golgi fraction with 0.5 M KC1 and 250 PM ATP and concentration by Diaflow. Backgrounds representing the incorporation in the absence of NSF and palmitoyl-CoA have been subtracted. These were 460 cpm (A) and 280 cpm (23). The bar in each pane l marked Control is the amount of incorporation when donor and acceptor that had not been treated with NEM were assayed.

restore transport to trans Golgi in a synergistic fashion. This suggests that these components may act at multiple levels of the Golgi stack.

DISCUSSION

Transport-coupled sialylation requires ATP, a cytosol fraction, NSF, and acyl coenzyme A and occurs under the same conditions in which we have previously found Golgi stacks to retain their integrity as measured both by their size and the number of cisternae they contain (Braell et al., 1984). Thus, it seems unlikely that the sialylation of G protein could result from unspecific membrane fusion or from fusion of the two populations of Golgi stacks. The rapid depletion of donor activity following cycloheximide treatment (Fig. 8) also argues against a nonspecific fusion. Rather, the straightforward interpretation of the sialylation reaction would be that G protein is transported from the donor stacks to the acceptor stacks during the incubation, as in the medial transport assay. Consistent with this interpretation is the finding that nonclathrin-coated vesicles bud from every cisterna in the Golgi stacks under these incubation conditions and contain G protein independent of the level at which they bud (Orci et al., 1986). The formal alternative, that the CMP-sialic acid permease missing from the clone 1021 donor Golgi is transported to these “donor” membranes while G protein remains static, cannot be ruled out at the present time. However, the movement of G protein to acceptor stacks has been demonstrated directly in the case of the medial assay by autoradiographic localization of the incorporated [ 3 H ] G l ~ N A ~ residues (Braell et al., 1984).

Where in the donor Golgi does the sialylated G protein begin its transit and where in the acceptor Golgi does it terminate? In CHO cells GlcNAc is added to G protein in a distinct and earlier compartment than either galactose or sialic acid (Rothman et al., 1984a, 1984b; Dunphy et al., 1981).

Based on analogy to results of electron microscope immuno- cytochemistry carried out in other types of cells (Roth and Berger, 1982; Roth et al., 1985 Dunphy et al., 1985) we have termed these successive functionally defined compartments in CHO cells “medial” and “trans.” The compartmental divi- sions that separate these glycosylation sites in CHO cells can and have been demonstrated by functional tests (Rothman et al., 1984a, 1984b) quite independently of any morphological evidence. Our earlier work with cell-free systems (Balch et al., 1984a) concerned the receipt of GlcNAc in uitro, and thus entry into the functional compartment defined as medial in CHO cells. Here we find that G protein that has received galactose in vivo goes on to acquire sialic acid in uitro. Since galactose is added in a functionally distinct and later compartment than GlcNAc (defined as trans) it is clear that with the sialylation assay we are measuring a different and later transport step than with transport-coupled incorporation of GlcNAc. This transit most likely involves movement between the functionally defined trans compartments of donor and acceptor stacks, defined as the sites of in vivo incorporation of galactose and sialic acid. If so, have we reconstituted a “lateral transfer” between cisternae at the same level of the Golgi stack? Or, are there possibly finer distinctions to be made between the sites of galactose and sialic acid incorporation within this functionally defined “trans” region of the Golgi, distinctions that would require another round of vesicular transport?

Very recent work has raised the possibility that the trans Golgi consists of two distinct compartments, both of which contain galactosyl- and sialyltransferases (Griffiths and Si- mons, 1986). The distinction between the two trans compartments is based primarily on their morphology in vivo and could offer an explanation of our results. In this model of four Golgi compartments (Griffiths and Simons, 1986), the fourth compartment is the trans-most Golgi cisterna, extensively


tubulated and containing many clathrin-coated buds as well as acid phosphatase in many cases. This terminal compartment has been termed the trans Golgi network (TGN). If trans Golgi (compartment 3) and TGN (compartment 4) are distinct and sequential as proposed (Griffiths and Simons, 1986), then a vesicular transport system would be needed to move transported proteins between them.

Given these considerations, transport-coupled sialylation may measure a reconstitution of transport of G protein from the trans compartment of the donor Golgi stack to the TGN of the acceptor Golgi stack.' This explanation suggests itself because the G protein starts out in the donor Golgi having already received galactose ( in uiuo) and then goes on to receive sialic acid during the cell-free incubation. However, it is difficult to be certain that this transport is directional in uitro. We cannot rule out that galactosylated G protein moves from the trans cisternae of the donor stack to earlier locations in the acceptor stack and then is transported through the acceptor stack to the sialylation site. However, there is no reason to believe that such retrograde movements exist in the Golgi stack (Rothman et al., 1984a, 1984b) nor does such a complex and indirect route seem very likely. I t is noteworthy that we observed dissociative transfers in which G protein labeled with [3H]galactose in VSV-infected 1021 cells moved to the Golgi of uninfected 15B cells to receive sialic acid after cell fusion (Rothman et al., 1984a). However, the probability of such a hopping was about five times lower than a hop from cis to medial or from medial to trans Golgi. The dissociative transfer of galactose-containing G protein, resulting in its sialylation in the cell fusion experiments seems most likely to represent the in vivo counterpart to the transport-coupled sialylation observed in uitro.

In summary, at least two segments of the pathway of protein transport in the Golgi stack have now been reconstituted. In the past we have reported the reconstitution of a segment that seems to connect cis to medial cisternae, resulting in a transport-coupled incorporation of GlcNAc (Balch et al., 1984a). Here, I have offered evidence that transport between trans Golgi elements occurs simultaneously and can be measured by a transport-coupled sialylation. Together with the observation that G protein-containing vesicles bud at all levels of the stacks in uitro (Orci et al., 1986), this suggests that the complete set of transport operations within the Golgi stack may be reconstituted together. It may be only a matter of

It is difficult to ascertain whether or not TGN is present in the Golgi fractions used as donor and acceptor since TGN does not as yet have a clearcut enzymatic marker to distinguish it from trans Golgi. Our Golgi fractions contain both galactosyl- and sialyltransferases in the same ratio as crude homogenates (Balch et al., 1984a). The extensive tubulation characteristic of TGN is observed after incubation of Golgi under transport conditions (Orci et al., 1986). However, the clathrin-coated pits that are also characteristic of TGN in vivo are not observed on Golgi incubated in uitro under our transport conditions (Orci et al., 1986). The transport conditions used were those that optimized intra-Golgi transport (Balch and Rothman, 1985) apparently mediated by non-clathrin-coated vesicles (Orci et al., 1986), so the lack of clathrin-coated vesicles may simply reflect that clathrin-coated pit formation has a different set of optimal conditions, rather than a lack of TGN in our preparation.

changing the window of observation in an appropriate way to reveal the occurrence of transfers at any level of the stack. The availability of assays that measure distinct and successive segments of transport in the Golgi stack should be of value by providing a test for the specificity of isolated transport components. For example, some proteins (like a coat protein) may be used at all levels of the stack, whereas other proteins (such as a vesicle-targeting protein) may be used at a single level only. Now such distinctions can be made with functional assays.

Acknowledgments--I thank Lyne Paquet for making membrane preparations, Michael Gleason for help with some experiments, Marc Block and Benjamin Glick for the NSF fraction, Stuart Kornfeld for suggesting the use of UDP-glucose dehydrogenase, and Felix Wieland for his criticism of the manuscript.

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