Cell. Vol 69

129-136.

April 3, 1992, Copyright

0 1992 by Cell Press

Recruitment of Coat Proteins onto Golgi Membranes in Intact and Permeabilized Cells: Effects of Brefeldin A and G Protein Activators Margaret S. Robinson” and Thomas E. Kreist *Department of Clinical Biochemistry University of Cambridge Addenbrookes Hospital Hills Road Cambridge CB2 2QR England tEuropean Molecular Biology Laboratory 6900 Heidelberg Federal Republic of Germany

Summary Brefeldin A (BFA) causes a rapid redistribution of coat proteins (e.g., y-adaptin) associated with the clathrincoated vesicles that bud from the Vans-Golgi network (TGN), while the clathrin-coated vesicles that bud from the plasma membrane are unaffected. y-Adaptin redistributes with the same kinetics as (J-COP, a coat protein associated with the non-clathrin-coated vesicles that bud from the Golgi complex. Upon removal of BFA, however, y-adaptin recovers its perinuclear distribution more rapidly. Redistribution of both proteins can be prevented by pretreating cells with AIF4-. Recruitment of adaptors from the cytosol onto the TGN membrane has been reconstituted in a permeabilired cell system and is increased by addition of GTPyS and blocked by addition of BFA. These results suggest a role for G proteins in the control of the clathrin-coated vesicle cycle at the TGN and further extend the similarities between clathrin-coated vesicles and nonclathrin-coated vesicles. Introduction Movement of proteins from one membrane compartment of the cell to another is accomplished by the budding and fusion of transport vesicles. The best characterized of these vesicles have been shown to be coated with proteins on their cytoplasmic sides. Binding of the coat proteins to the appropriate membrane is thought to be a prerequisite for budding, while uncoating of the vesicle appears to be required for fusion. Two types of coated vesicles have so far been identified: clathrin-coated vesicles and non-clathrin-coated, or COPcoated, vesicles. The coats on clathrin-coated vesicles consist of two protein complexes, clathrin and adaptors, which exist in the cytoplasm as separate soluble pools (Pearse and Robinson, 1990). Clathrin plays a mechanical role in vesicle budding, while adaptors are thought to attach the clathrin to the cytoplasmic domains of selected membrane proteins such as the LDL receptor and the mannose-g-phosphate receptor (Pearse, 1988; Glickman et al., 1989). There are two subsetsof clathrin-coated vesicles in the cell, one associated with the plasma membrane and one associated with the Vans-Golgi network (TGN),

which recruit different types of adaptors (Robinson, 1987; Ahle et al., 1988). Both adaptors are heterotetramerscomposed of related subunits: the plasma membrane adaptor consists of a-adaptin, l3-adaptin, a 50 kd subunit, and a 17 kd subunit, while the Golgi adaptor consists of y-adaptin, o’-adaptin, a 47 kd subunit, and a 20 kd subunit. These differences are believed to account for the ability of the two subsets of clathrin-coated vesicles to recognize and concentrate different membrane proteins. However, it is thought unlikely that the binding of adaptors to such membrane proteins is the way in which the adaptors are targeted to the plasma membrane or the TGN, since proteins like the LDL receptor and the mannose-6-phosphate receptor are found in other membrane compartments as well as those with which adaptors are associated. Thus, there may be another mechanism for recruiting adaptors to the appropriate membrane initially, after which they can bind to the cytoplasmic domains of proteins in the same compartment. The coat proteins (COPS) of non-clathrin-coated vesicles are less well characterized than those of clathrincoated vesicles, since these vesicles were first isolated only 2 years ago (Malhotra et al., 1989). However, recently four COPS (a, b, y, and 6) have been identified, which are found both on the isolated coated vesicles and as a cytosolic complex (Serafini et al., 1991; Waters et al., 1991; Duden et al., 1991). The COP-coated vesicles are thought to mediate the bulk flow of newly synthesized proteins through the Golgi stack, without actually concentrating such proteins (Orci et al., 1986). In spite of the differences between the two types of coated vesicles, structural as well as functional, recent studies have shown that B-COP shares sequence homology with the adaptor subunits b-and P’-adaptin (Duden et al., 1991). Moreover, the similarity in size of some of the other subunits of clathrincoated and COP-coated vesicles suggests that there may be additional homologies (Malhotra et al., 1989; Serafini et al., 1991). Thus, it seems likely that the two types of coated vesicles form by a similar mechanism. The cycling of coat proteins on and off the membrane is an important control step in vesicular traffic, but little is known about how these cycles are regulated. A system has been developed for studying the binding of clathrin and adaptors to the plasma membranes of permeabilized cells, and progress is being made toward identifying a binding site(s) on the membrane (Mahaffey et al., 1990). However, because it is first necessary to remove existing coat proteins with nonphysiological treatments such as high concentrations of Tris-HCI or salt, it is still not known how coated pits are initiated in vivo. Other studies using permeabilized cells have shown that addition of cytosol and ATP results in the formation of new coated pits on the plasma membrane, although it is not clear whether coat proteins are being recruited from the cytosol, or whether such proteins were already bound to the membrane and are being induced to assemble (Smythe et al., 1989). So far, no equivalent work has been carried out on the

Cell 130

p-cop

o-adaptin

y-adaptin

clathrin

Figure 1. Distribution treated Vero Cells

of Coat Proteins

in Un-

Vero cells were stained with mouse MAbs against B-COP (a), a-adaptin (b). v-adaptin (c), and clathrin heavy chain (d) and double labeled with a rabbit antiserum against galactosyltransferase (e-h). Bar = 10 pm.

clathrin-coated vesicles associated with the TGN. The uncoating step has also been studied in a cell-free system, leading to the finding that a member of the family of heat shock proteins, hsc70, removes clathrin from coated vesicles in vitro (Schlossman et al., 1984; Chappell et al., 1986). However, because hsc70onlyremovesclathrin and not adaptors, there are clearly other uncoating factors that have not yet been isolated. Different approaches have been used to investigate the association of COPS with the Golgi membrane, based on in vitro reconstitution studies of membrane traffic through the Golgi stack (Balch et al., 1984). Addition of cytosol and ATP to isolated Golgi stacks results in the budding of COP-coated vesicles, which can be stabilized by addition of GTPyS (Orci et al., 1989) or prevented from forming by addition of the drug brefeldin A (BFA) (Orci et al., 1991). In living cells, BFA causes a rapid redistribution of p-COP away from the Golgi complex so that it appears diffuse throughout the cytoplasm within less than 1 min, as assayed by immunofluorescence (Donaldson et al., 1990). More prolonged treatments with BFA result in breakdown of the Golgi stack and return of resident Golgi proteins to the endoplasmic reticulum (ER) (Lippincott-Schwartz et al., 1989; Doms et al., 1989). Because the COPS were found to be the proteins affected earliest by the drug, it has been suggested that their redistribution is responsible for the changes that occur later (Donaldson et al., 1990). All of these changes can be prevented by pretreating intact cells with AIFd- or by pretreating permeabilized cells with GTPyS (Donaldson et al., 1991a), both of which are thought to act by locking G proteins in the active (i.e., GTP-bound) state. The model that has been proposed to explain these findings is that BFA blocks the coating step, while G protein activators block the uncoating step (Donaldson et al., 1991a; Orci et al., 1991). Because of the similarities now known to exist between

clathrin-coated and COP-coated vesicles, we have looked at the effects of BFA and AIF,- on the clathrin-coated vesicle cycle. The results of these studies have led to the development of a permeabilized system for reconstituting clathrin-coated vesicle formation at the TGN, and in particular for studying the targeting of adaptors to the TGN membrane. Results Redistribution of Coat Proteins in Response to BFA To study the effects of BFA on the distribution of coat proteins, Vero cells were incubated with the drug at a concentration of 5 pglml for various lengths of time and then examined by immunofluorescence microscopy. Figure 1 shows control cells stained with mouse monoclonal antibodies (MAbs) against p-COP, a-adaptin, y-adaptin, and clathrin (a-d) and double labeled with a rabbit antiserum against galactosyltransferase, an integral membrane protein resident in transQolgi cisternae (e-h). @COP primarily colocalizes with galactosyltransferase, although some punctate staining of Golgi-derived vesicles can also be seen. This is the typical pattern for P-COP that has been seen in a number of different cell types. The antibody against a-adaptin, a component of the plasma membrane adaptor complex, stains numerous dots corresponding to endocytic clathrin-coated pits and vesicles and does not show any coincidence with the Golgi marker. y-Adaptin is a component of the Golgi adaptor complex and is found in the same vicinity as galactosyltransferase. However, because the protein is associated with the TGN rather than with the entire Golgi complex, the labeling is less coincident than that observed with antibodies against P-COP. Moreover, there is also labeling of structures that are some distance away from the perinuclear region of the cell, which may represent either coated vesicles that have

Recrurtment 131

b-COP

of Coat

Proterns

onto Golgi

cc-adaptin

Membranes

y-adaptin

clathrin

Figure

2. Effects

of BFA on the Distribution

of

Vero cells were treated with 5 pglml BFA for 2 min at 37%. then stained with mouse MAbs against B-COP (a), a-adaptin (b), v-adaptin (c). and clathrin heavy chain (d), and double labeled with an antiserum against galactosyltransferase (e-h). A complete redistribution of both p-COP and y-adaptin can be seen, although the galactosyltransferase labeling is unaffected at this time point. Labeling with anti-clathrin shows that the clathrin associated with the Golgi complex has redistributed, but not the clathrin assoctated with the plasma

moved away from the Golgi complex or coated vesicles associated with more peripheral elements of the TGN or with some other compartment. The anti-clathrin staining looks like the anti-a-adaptin and anti-y-adaptin staining combined, as expected since the antibody labels both subsets of clathrin-coated vesicles. After the cells have been incubated for 2 min with BFA (Figure 2) the distribution of some of the coat proteins has changed, although the galactosyltransferase labeling still looks essentially normal. 6COP has completely redistributed at this time point, as previously reported (Donaldson et al., 1990). Furthermore, y-adaptin also shows a dramatic redistribution, and examination of earlier time points indicates that the two proteins dissociate from the Golgi with exactly the same kinetics. In contrast, the distribution of a-adaptin appears to be unaffected by the drug. Clathrin again looks like a combination of a- and y-adaptin, indicating that it is no longer associated with the TGN but is still associated with the plasma membrane. Longer incubations with the drug induce a redistribution of galactosyltransferase, with similar kinetics to those reported by others for integral membrane proteins of the Golgi stack (Lippincott-Schwartz et al., 1989; Doms et al., 1989) while the distribution of the coat proteins remains essentially the same as at the 2 min time point. Even after a 1 hr incubation with BFA, a-adaptin labeling appears unchanged (not shown). Thus, these results indicate that the drug specifically affects coat proteins associated with the Golgi complex, without changing the distribution of those associated with the plasma membrane. Although both 6COP and r-adaptin appear by immunofluorescence to be evenly distributed throughout the cytoplasm after BFA treatment, cell fractionation and sucrose sedimentation experiments did not reveal a significant increase in the soluble fraction of either protein. However, it is worth noting that both proteins have a grainy appear-

ance in drug-treated cells (see Figures 2a and 2c and Figures 3c and 3d), rather than the uniformly diffuse appearance typical of soluble proteins (see for example Kreis et al., 1979). Thus, it is possible that cytoplasmic coat proteins aggregate after BFA treatment. Recovery after BFA Wash-Out To compare the fates of p-COP and y-adaptin upon removal of BFA, Vero cells were examined at different time points after wash-out of the drug by double labeling with a rabbit anti+COP antibody raised against a synthetic peptide and a mouse MAb against y-adaptin. Figure 3 shows the two staining patterns in control cells (a and b) and in cells treated with BFA for 2 min (c and d). The cells in Figures 3e-3h were treated with BFA for 30 min, after which the drug was removed and the cells were allowed to recover for 2 (e and f) or 60 min (g and h). The rate of reassociation of f3COP with the reforming Golgi complex was found to be similar to that reported for NRK cells (Donaldson et al., 1990). Distinct Golgi patches, positive for galactosyltransferase as well as P-COP, can be detected after 20-30 min (not shown), and after 1 hr normal Golgi labeling can be seen (a and g). In contrast to B-COP, y-adaptin very rapidly (within l-2 min) reassociateswith tubular structures in the perinuclear region (Figure 3f). Similar tubular structures have recently been described in studies on the effects of BFA on membrane proteins of the TGN and endosomes, including TGN 38 (Lippincott-Schwartz et al., 1991; Reaves and Banting, 1992) and the mannose6-phosphate receptor (Wood et al., 1991). However, double labeling studies indicate that the tubules that stain for y-adaptin are for the most part negative for both of these proteins (data not shown), suggesting that y-adaptin may be associating with a special subcompartment of the TGN. One hour after BFA washout, y-adaptin has regained its normal distribution (Figures

Cell 132

give essentially full protection in NRK cells, although interestingly, relatively strong protection was seen fory-adaptin in these cells (data not shown). To examine the effect of AIFI- pretreatment on y-adaptin distribution in NRK cells, it was necessary to transfect the cells with a construct that reacts with y-adaptin antibodies (Robinson, 1990). This construct was prepared by splicing together y-adaptin sequences from mouse and cow, so that the epitope recognized by MAb 100/3 (Ahle et al., 1988) would be present. Previous studies have shown that the construct behaves like normal y-adaptin when transfected into Rat 1 fibroblasts (Robinson, 1990). When expressed in control NRK cells or in NRK cells that were treated with AIF4- alone (Figure 4), the chimeric y-adaptin (a and b) shows typical perinuclear and punctate labeling, coinciding to some extent with P-COP (e and 9 but also with a concentration of dots nearer to the cell periphery. BFA addition causes redistribution of both (j-COP and the y-adaptin construct to an apparently cytoplasmic location (c and g). However, pretreatment with AIF,- has a strong protective effect on both proteins (d and h). Thus, although AIF,- pretreatment varies from one cell type to another in its ability to protect them from subsequent addition of BFA and also varies in its ability to stabilize the two types of coat proteins within the same cell, in NRK cells it has a similar effect on both p-COP and y-adaptin.

Figure 3. Reassociation ing BFA Wash-Out

of Coat Proteins

with the Golgi Complex

dur-

Control Vero cells (a and b) or cells treated for 2 min (c and d) or 30 min (e-h) with 5 uglml BFA, then transferred back to normal culture medium for 0 (c and d), 2 (e and 9, or 60 min (g and h), were fixed and double labeled with a rabbit antibody against p-COP, anti-EAGE (a, c, e, g) and a mouse MAb against y-adaptin, 100/3 (b, d, f. h). Mitotic cells in metaphase can be seen in (a)-(d). In untreated mitotic cells, both coat proteins haveapatchydistribution, indicating thattheyareassociated with Golgi remnants (arrows), while after BFA treatment these patches disappear and the staining takes on the uniformly grainy appearance also seen in interphase cells. Two minutes after BFA washout, most of the y-adaptin has reassociated with tubular structures extending away from a perinuclear focus (arrows in [f]). No accumulation of 5COP can be detected in this perinuclear region (arrowheads in [e] and [f]). Bar = 20 urn.

3b and 3h). Thus, the effect of the drug upon y-adaptin, like its effect upon other proteins, is fully reversible. Protection by AIF4The BFA-induced redistribution of 6COP in NRK cells can be prevented by pretreating thecellswith AIF,-(Donaldson et al., 1991a). However, because NRK cells are derived from rat, they cannot be labeled with anti-y-adaptin antibodies, since to date no such antibodies have been raised that cross-react with the rodent protein. Thus, the effect of AIF4- pretreatment was investigated in Vero, muntjac, and COS cells. In all cases, staining with antibodies against B-COP showed only slight protection under conditions that

Targeting of Adaptors in a Permeabilized Cell System To study factors involved in the regulation of the clathrincoated vesicle cycle more fully, a permeabilized cell system was developed that would reconstitute the targeting of adaptors from the cytosol to the TGN membrane, making use of the species-specific MAb 10013. In this system, acceptor NRK cells are grown on multiwell glass slides or in dishes, permeabilized by rapid freezing and thawing, and incubated with donor cytosol prepared from human HL-60 cells. Because MAb 10013 only recognizes y-adaptin from the donor cells (human) and not from the acceptor cells (rat), membrane association of the protein can be assayed by immunofluorescence or Western blotting without any background from endogenous y-adaptin. Figure 5 shows the results of an immunofluorescence experiment, in which the cells were double labeled with MAb 10013 (a-d) and an antiserum against TGN 38 (Luzio et al., 1990) (e-h). Acceptor cells that were incubated with control donor cytosol (a) show faint but distinct perinuclear staining with the antibody against y-adaptin. Such staining is absent in control NRK cells (see for example the nontransfected cells in Figure 4). Double labeling with the antiserum against TGN 38 (Figure 5e) confirms that the y-adaptin has been targeted to the correct intracellular location. When ATP, GTP, and an ATP regenerating system are added to the donor cytosol (Figures 5b and 5f), a marked increase in staining can be seen with MAb 100/3, indicating that targeting of Golgi adaptors to the TGN compartment is nucleotide dependent. Addition of GTPyS, which has been shown to stabilize the coats on COP-coated vesi-

Recrurtment 133

of Coat

control

Proteins

onto Golgi

AIF4-

Membranes

BFA

cles, results in even stronger labeling (c and g). In addition, ATPrS also results in stronger labeling than that seen with hydrolyzable nucleotides (d and h). Effect of BFA on Adaptor Targeting If BFA acts by preventing coat proteins from binding to the Golgi membrane, as has been proposed for p-COP, then addition of the drug to the donor cytosol should prevent y-adaptin from associating with theTGN, even with nucleotides and a regenerating system. Addition of 10 ug/ml BFA results in decreased labeling (not shown), while addition of 100 uglml BFA completely abolishes labeling associated with the TGN (Figure 6a). The distribution of TGN 38 is

control

ATP + GTP

GTPyS

AIF& + BFA

Figure

4. Prevention

of BFA-Induced

Redistri-

also affected by the drug (Figure se): tubular extensions can be seen, similar to those induced by BFA in vivo (Lippincott-Schwartz et al., 1991; Reaves and Banting, 1992). Although 100 uglml is considerably higher than the concentration of BFA required for studies on intact cells, others have also found that the drug is less effective in vitro than in vivo (Orci et al., 1991). Two lines of evidence indicate that the effect of BFA is a specific one rather than a side effect due to the high concentrations of the drug. First, addition of 100 uglml of a relatively inactive analog of BFA, B16, has no effect on adaptor targeting (Figures 6b and 6f; see Figures 5b and 5f for comparison). Second, pretreatment of the permeabilized cells with cytosol plus

ATP$

Figure

5. Effects

of Nucleotides

on Adaptor

Permeabilized NRK acceptor cells were rncubated for 10 min at 37°C with donor HL-60 cell cytosol (with or without additions), then double labeled with the species-specific anti-yadaptin MAb 100/3 (a-d) and a rabbit antiserum against TGN 36 (e-h). (a) and (e) show cytosol with no additions. (b) and (9 show cytosol plus 1 mM ATP, 1 mM GTP, and an ATP regenerating system (10 mM phosphocreatine and 20 U of creatine kinase). (c) and (g) show cytosol plus 1 mM GTPyS. (d) and (h) show cytosol plus 1 mM ATPrS. The cell on the left in (c) and (g) was poorly permeabilized. and thus only reacts with the anti-TGN 36 antibody. Bar = 10 pm.

Cell 134

BFA

B16

BFA GTPyS

GTPyS BFA

Figure 6. Effects of BFA and a BFA Analog on Adaptor Targeting Permeabilized NRK acceptor cells were incubated at 37% with donor HL-60 cell cytosol plus various reagents, then double labeled with the species-specific anti-padaptin MAb 10013 (a-d) and a rabbit antiserum against TGN 38 (e-h). (a) and (e) show 10 min incubation with cytosol plus 1 mM ATP, 1 mM GTP, an ATP regenerating system, and 100 uglml BFA. (b) and (9 show 10 min incubation with cytosol plus 1 mM ATP, 1 mM GTP, an ATP regenerating system, and 100 pglml 816. (c) and (g) show 10 min incubation with cytosol plus 1 mM ATP, an ATP regenerating system, and 100 uglml BFA, followed by another 10 min incubation with the above reagents plus 1 mM GTP+. (d) and (h) show 10 min incubation with cytosol plus 1 mM ATP, an ATP regenerating system, and 1 pM GTPTS, followed by another 10 min incubation with the above reagents plus 100 uglml BFA. Bar = 10 urn.

GTPTS protects them from subsequent addition of BFA, even at 100 uglml (Figures 6d and 6h). In contrast, if the permeabilized cells are first treated with cytosol plus BFA and then with GTPyS, no TGN-associated labeling is seen (Figures 6c and 6g). Similarly, the order of addition of BFA and GTPTS has also been shown to be important in studies on the redistribution of P-COP in permeabilized cells, and this observation has been cited as evidence for the model that BFA acts by preventing membrane coating rather than by inducing membrane uncoating (Donaldson et al., 1991a). Targeting Assayed by Western Blotting The advantage of immunofluorescence as an assay for y-adaptin targeting is that it demonstrates that the protein is specifically binding to the TGN rather than associating nonspecifically with other parts of the cell. However, the drawback of such an assay is that it is difficult to quantify, especially in permeabilized cells where the degree of permeabilization may vary from one cell to another. Thus, a second assay was developed that involved permeabilizing a larger sample of cells, which were then divided into aliquots and incubated with cytosol plus various reagents before they were pelleted at low speed, washed, and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting. Figure 7 shows a typical Western blot, while Figure 8 quantifies the signal produced by y-adaptin under various conditions, using data pooled from three separate experiments. Addition of GTPyS to the donor cytosol (Figure 7, lane 5P) results in a -lo-fold increase in y-adaptin radioactivity pelleting with the acceptor cells when compared with cells incubated with cytosol alone (Figure 7, lane 2P; Figure 8). Examination of thesupernatants(Figure 7, lanes 2s and 5s) indicates that much of the y-adaptin in the

donor cytosol has been recruited to the acceptor cells in the presence of GTPyS. Addition of ATPTS also results in increased label associated with the pellet (lane 4P), although the effect is less than that observed with GTPyS (see Figure 8). Pretreatment with BFA before addition of GTPyS largely prevents r-adaptin from pelleting with the acceptor cells (lane 6P), while pretreatment with GTPyS protects the acceptor cells from subsequent addition of the drug (lane 7P). These results are all consistent with the immunofluorescence observations. Unexpectedly, however, addition of ATP, GTP, and an ATP regenerating system to the cytosol actually causes a reproducible decrease in the amount of signal pelleting with the acceptor cells (lane 3P) when compared with cytosol alone (lane 2P). In contrast, immunofluorescence experiments show an increase in Golgi-associated y-adaptin under these conditions (see Figure 5). Although we do not as yet have an explanation for this discrepancy, it seems to be related to the more extensive washing conditions that are required when preparing cells for Western blotting. It is possible that nucleotides may be promoting the budding of coated vesicles that are then washed away or that nucleotidedependent uncoating may be occurring during the wash step. Thus, the lability of the association of y-adaptin with the Golgi complex in the presence of nucleotides may reflect the dynamic nature of the coated vesicle cycle. Discussion We have demonstrated that the effect of BFA on clathrincoated vesicles associated with the TGN is similar to its effect on COP-coated vesicles. This result further highlights the similarities between the two types of coated vesicles, first suspected because of the similar sizes of some of their coat proteins (Malhotra et al., 1989) and later con-

Aecrurtment 135

of Coat

IP

Proteins

2P

3P

onto Golgi

4P

5P

6P

Membranes

7P

cyt 2s 5s no add!tions

ZOOK

ATP + GTP

116K

-

97K

-

66K

-

45K

-

ATP~fi

BFA i GTPyS

GTPyS

/ BFA

10

20

yadaptin Figure 13. Quantification Donor Cytosol

Figure

7. Biochemical

Assay

for Adaptor

Targeting

NRK cellswere incubated with HL-60 cell donor cytosol (or buffer), then centrifuged at low speed. Pellets and supernatants were subjected to SDS-PAGE and Western blotting and probed with the species-specific anti-y-adaptin MAb 10013, followed by goat anti-mouse IgG and ‘%labeled protein A. Lanes IP-7P are pellets of cells incubated for IO min at 37OC under the following conditions. 1P: buffer alone. 2P: cytosol without additions. 3P: cytosol plus 1 mM ATP, 1 mM GTP, and an ATP-regenerating system. 4P: cytosol plus 1 mM ATP$. 5P: cytosol plus 1 mM GTPyS. 6P: cytosol plus 1 mM ATP, an ATPregenerating system, and 100 uglml BFA, followed by 10 min in cytosol containing the above reagents plus 1 mM GTPTS. 7P: cytosol plus 1 mM ATP, an ATP-regenerating system, and 1 mM GTPyS, followed by 10 min in cytosol containing the above reagents plus 100 pglml BFA. Lane “cyt”: donor cytosol. Lane 25: supernatant from the cells in Lane 2P. Lane 5s: supernatant from the cells in lane 5P. The y-adaptin band is just below the 97 kd standard. Other radiolabeled bands are also seen on blots from which the primary antibody was omitted and thus are nonspecific.

firmed by the sequence homology between p-COP and @adaptin (Duden et al., 1991). Interestingly, however, the clathrin-coated vesicles associated with the plasma membrane appear to be resistant to the drug. We also find other similarities between the two types of Golgi coat proteins: both redistribute in response to energy depletion as well as BFA (T. E. K., unpublished data), and both can be protected from BFA-induced redistribution if the cells are pretreated with AIF,-. Moreover, in permeabilized cells, GTPyS promotes the association of both types of Coat proteins with the appropriate membrane and protects them from subsequent addition of BFA (see Donaldson et al., 1990, 1991 a, 1991 b). The use of permeabilized cells should allow the molecular basis of these events to be dissected, since we can now add defined components to the system. Previous studies on BFA-treated cells have shown that the drug induces a rapid redistribution of 8COP away from the Golgi region, followed by a slower redistribution of Golgi membrane proteins so that they mix with the ER

30

40

50

signal

of the Signal Produced

by y-Adaptin

from the

Blots similar to the one shown in Figure 7, but with more widely separated lanes, were prepared from threeseparateexperiments. The autoradiographs were scanned on a densitometer, and the areas under the u-adaptin peaks in lanes 2P-7P were measured. Numbers are expressed as percent signal from all six lanes; 2P through 7P are shown from top to bottom. The error bars show the standard deviations.

(Lippincott-Schwartz et al., 1989; Donaldson et al., 1990). Because the redistribution of p-COP was the earliest change to be detected in response to the drug, it was suggested that COPS might be direct targets of BFA and that their dissociation from the Golgi membrane might be the cause of the subsequent changes observed in the Golgi stack (Donaldson et al., 1990). The experiments reported in this article demonstrate that BFA causes an equally rapid redistribution of adaptors away from the TGN, indicating either that Golgi adaptors are also direct targets of the drug, or that BFA is acting upstream of both. The second possibility seems more likely for at least two reasons. First, the homology between the COPS and the adaptor subunits appears to be relatively weak (Duden et al., 1991). In contrast, the plasma membrane adaptors, which are much more closely related to the Golgi adaptors (Robinson, 1990; Kirchhausen, 1991) are unaffected by the drug. Second, there are other reagents in addition to BFA that have similar effects on the coat proteins of clathrin-coated and COP-coated vesicles, including energy blockers (T. E. K., unpublished data), AIF,-, and GTPrS (see Donaldson et al., 1991a, 1991 b). Thus, it is likely that the two coated vesicle cycles are regulated by a similar mechanism that is sensitive to BFA. The AIFlm results suggest that heterotrimeric G proteins are likely to be involved in both cycles, possibly by sending signals across the Golgi membrane that induce the cell to recruit cytoplasmic proteins for making a particular type of vesicle. Although in most ways the effects of BFA on the distribution of 8COP and v-adaptin are identical, we also find some differences between the two proteins. First, in our BFA wash-out experiments, y-adaptin recovers its perinuclear distribution at a time when B-COP still appears to be cytoplasmic (see Figure 3). This may be a reflection of the

Cell 136

distribution of the various Golgi subcompartments after prolonged treatments with the drug, although since there is evidence that p-COP is associated with the TGN as well as with the Golgi stack (Duden et al., 1991; G. Griffiths and T. E. K., unpublished data), one would expect at least some of the P-COP to colocalize with the v-adaptin. It is possible that there may be subdomains of the TGN which recruit different coat proteins and which recover from BFA at different rates or that (3-COP cannot associate with the TGN in the absence of a fully assembled Golgi complex. Other differences between the two proteins include the findings that pretreatment with AIF4- has a somewhat stronger protective effect on y-adaptin than on j3COP in three cell lines and that a less active analog of BFA, 827, needs to be used at a 1OO-fold higher concentration in Vero cells to induce redistribution of y-adaptin than to induce redistribution of j3COP (M. S. R., unpublished data). In addition, D. Wong and F. Brodsky, who have independently carried out studies similar to ours, have found that in PtKl cells, y-adaptin distribution is affected by BFA, while f3COP distribution is unaffected (F. Brodsky, personal communication). These results suggest that the effects of BFA on COPS and adaptors may be mediated through related but distinct molecules, such as organellespecific G proteins, consistent with recent reports that different membrane compartments vary in their susceptibility to BFA from one cell line to another (Ktistakis et al., 1991; Hunziker et al., 1991; Sandvig et al., 1991). It has been proposed that BFA interferes with the COPcoated vesicle cycle by preventing cytoplasmic COPS from binding to the Golgi membrane, rather than by stripping COPS from the membrane once they have bound (Donaldson et al., 1991a; Orci et al., 1991). Thus, according to this model, in the presence of the drug, cells would be able to complete one COP-coated vesicle cycle but would be unable to start the next. Evidence in support of the model comes from the finding that reagents such as AIF4- and GTPyS can block the effects of BFA on P-COP only if they are added first. Because we have obtained similar results in our studies on y-adaptin, it seems likely that BFA prevents the association of both types of coat proteins with the appropriate membrane. However, the drug acts much more quickly than the time thought necessary to carry out one coated vesicle cycle. Moreover, BFA has a rapid effect on the distribution of coat proteins in mitotic cells as well as interphase cells (see Figures 3a-3d), causing both f3COP and y-adaptin to acquire a uniform rather than patchy distribution within 2 min; yet vesicular traffic is thought to stop during mitosis (Warren, 1989). Thus, it is possible that BFA may also be inducing coat proteins to dissociate from the membrane, but that the proteins are somehow stabilized by AIF,- or GTPyS. Another possibility is that the association of coat proteins with the Golgi complex may in fact be a highly dynamic one, in which the proteins repeatedly cycle on and off the membrane before they are incorporated into a budding vesicle. This possibility is consistent with the recent resultsof Davoust and Cosson (1991), who microinjected cells with fluorescent clathrin and found an unexpectedly rapid recovery of fluorescence after photobleachrng (tM = 11 s), thought to reflect the rate of ex-

change between membrane-bound and soluble clathrin. Thus, present models of coated vesicle cycles may have to be modified to include a rapid cycle of coat protein association/dissociation as well as the slower cycle of vesicle budding and uncoating. Recent studies have shown that the effects of BFA are more widespread than was originally believed. It is now clear that not only the Golgi stack, but also the TGN, endosomes, and lysosomes send out long tubular extensions in response to the drug, while normal vesicular traffic between these compartments is impeded (LippincottSchwartz et al., 1991; Wood et al., 1991; Hunziker et al., 1991; Sandvig et al., 1991; Reaves and Banting, 1992). When these observations were reported, it was proposed that there might be other BFA-sensitive coat proteins associated with these organelles, similar to but distinct from the COPS, and that their dissociation could be the cause of the subsequent disruption in post-Golgi membrane traffic. The Golgi adaptors and clathrin are obvious candidates for such proteins, although there are also likely to be additional coat proteins that have not yet been identified. Loss of such coat proteins from the TGN, endosomes, and lysosomes could prevent the normal shuttling of vesicles between these organelles and lead to a breakdown in traffic control, just as loss of COPS is thought to interfere with normal traffic through the Golgi stack. Although much remains to be learned about the way in which BFA acts, the drug should prove a valuable tool for elucidating factors involved in the binding of coat proteins to membranes during both the clathrin-coated vesicle cycle and the COP-coated vesicle cycle. The permeabilized cell system described in this article should also aid in our understanding of these events. Preliminary immuno-EM results indicate that adaptors recruited from the donor cytosol are not only able to bind to the TGN membrane, but are also able to become incorporated into clathrin-coated buds. It is not yet clear whether we are able to reconstitute the entire cycle with this system; however, one possible explanation for the lability of the association of y-adaptin with acceptor cells in the presence of nucleotides and a regenerating system is that the coated buds are pinching off as coated vesicles and are then becoming uncoated. Omitting the nucleotides or adding their nonhydrolyzable analogs could lead to a rigor-like situation, in which the cycle has been blocked at a particular point. GTPyS and ATPyS may be interfering with the cycle at different stages. Indeed, both GTP and ATP probably act at more than one step. Recent work has shown that there are several different types of GTP-binding proteins involved in vesicular traffic: heterotrimeric G proteins have been implicated as well as monomeric G proteins belonging to the ras superfamily and the dynamin family (see Stow et al., 1991; Balch, 1990; Collins, 1991). There are likely to be multiple steps at which ATP is required as well (Smythe et al., 1989; Schlossman et al., 1984). Thus, nonhydrolyzable nucleotide analogs should be useful probes for dissecting different stages of the clathrin-coated vesicle cycle in the permeabilized cell system. This system is still relatively crude, but it should be possi-

Recruitment 137

of Coat Proteins

onto Golgi

Membranes

ble to refine it by fractionating both the donor cytosol and the acceptor cell membranes. In addition, we hope to develop similar systems for studying the targeting both of COPS and of plasma membrane adaptors, using antigenitally tagged coat proteins. It is clear from the experiments reported in this article that there are a number of important differences between the clathrin-coated vesicle cycle at the TGN and the one at the plasma membrane, consistent with the finding that endocytosis still carries on normally in the presence of BFA (Lippincott-Schwartz et al., 1991). Nevertheless, given the degree of homology between the two adaptor complexes, there are also likely to be important similarities in the way they cycle on and off their respective membranes. The plasma membrane adaptors, the Golgi adaptors, and the COPS are just some of the many proteins and protein complexes involved in vesicular traffic that must be precisely targeted to a particular compartment and that must repeatedly cycle back and forth between membrane and cytoplasm. Although these processes are still poorly understood, it is possible that there may be some common features in the way they are regulated throughout the cell. Experimental

Procedures

Reagents and Antibodies BFA was obtained from Epicenter Technologies (Madison, WI) and stored frozen as a 5 mglml stock solution in ethanol or dimethyl sulfoxide. Analogs of the drug were kindly donated by Rick Klausner (NIH, Bethesda, MD). Antibodies used for immunolabeling included MAb 10013, a mouse MAb against y-adaptin (Ahle et al.. 1988); M3A5, a mouse MAb against p-COP (Allan and Kreis, 1986); anti-EAGE, an affinity-purified rabbit polyclonal antibody against an 18 residue peptide derived from B-COP (Duden et al., 1991); ACI-Mll, a mouse MAb against a-adaptin (Robinson, 1987); X-22, a mouse MAb against clathrin heavy chain (Brodsky, 1985); a rabbit polyclonal antibody against galactosyltransferase (Roth and Berger, 1982); and a rabbit polyclonal antibody against TGN 38 expressed as a fusion protein in Escherichia coli (Luzio et al., 1990). Cells Cell lines that were used in this study included Vero cells, NRK cells, and HL-60 cells. They were all grown in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, to which BFA and other reagents were added for in vivo experiments. In BFA wash-out experiments, cells were extensively washed in normal culture medium and then incubated in that medium for various lengths of time. Fixation and preparation for immunofluorescence were carried out essentially as previously described (Robinson, 1987; Duden et al., 1991). For some experiments, NRK cells were transfected by the calcium phosphate coprecipitation method with plasmid pHYKS4 (Robinson, 1990). This plasmid contains a cDNA insert encoding a chimeric y-adaptin, constructed from mouse and cow sequences and containing the species-specific epitope recognized by MAb 100/3, downstream of the SV40 early promoter. Cells were treated and prepared for immunofluorescence 48 hr after transfection to maximize transient expression. Adaptor Targeting in Permeabilized Cells For immunofluorescence studies, NRK cells were grown on poly-Llysine-coated multiwell test slides and used when they were ~50% confluent. To prepare the cells for permeabilization, the slides were washed with cytosol buffer (25 mM HEPES-KOH [pH 7.01, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM dithiothreitol, and 1 mg/ml glucose) (Donaldson et al., 1991a) and dried by blotting and aspiration, leaving only residual buffer over the wells. The cells were then frozen by placing the slides on a metal block that had been cooled on dry ice. After thawing, the wells were rinsed with cytosol buffer and incubated with 12 ~1 drops of cytosol for 10 min at 37%.

Experiments that involved pretreatment were carried out by incubating the permeabilized cells for 10 min at 37OC with cytosol containing the first reagent, after which the cytosol was replaced with fresh cytosol containing the second reagent(s). The cells were then washed with cytosol buffer, fixed with methanol and acetone, and prepared for immunofluorescence. NRK cells to be used for Western blotting were grown until just confluent in 9 cm tissue culture dishes. They were washed with cytosol buffer and then frozen by floating the dish briefly in liquid nitrogen. After thawing, the permeabilized cells were scraped up with a rubber policeman and harvested by centrifugation for 30 s at low speed (6500 rpm) in a microfuge. Cells from two dishes were typically divided into seven aliquots. washed with cytosol buffer, and incubated with 50 ~1 of buffer or cytosol for 10 min at 37% They were then spun again at low speed, washed with cytosol buffer, and boiled with sample buffer for SDS-PAGE. Equal volumes of the resuspended pellets were loaded into each of the wells. For some experiments, aliquots of the supernatants were also boiled with sample buffer and subjected to electrophoresis and Western blotting. Blots were probed with MAb 100/3, followed by rabbit anti-mouse immunoglobulin G (IgG) (Sigma Chemical Company, St. Louis, MO) and ‘%protein A (New England Nuclear, Boston, MA) as previously described (Robinson, 1987). Cytosol was prepared from HL-60 cells that had been grown in 500 ml roller bottles, harvested bycentrifugation, and washed with cytosol buffer. The packed pellet was then resuspended in an equal volume of cytosol buffer, and the cells were disrupted by freezing in liquid nitrogen and thawing. Complete disruption was achieved by aspirating the frozen and thawed cells up and down 20 times using a 19 gauge needle. Cytosol was obtained by centrifuging the cells for 15 min at 50,000 rpm in a benchtop ultracentrifuge and was frozen in aliquots in liquid nitrogen until required. The protein concentration of the cytosol was -16 mglml. Acknowledgments Darren Wong and Frances Brodsky independently carried out similar experiments on the effects of BFA on clathrin-coated vesicles at the same time that we did. We thank them for communicating their unpublished data. We are extremely grateful to Ernst Ungewickell for supply ing the anti-r-adaptin MAb 10013, without which these studies could never have been carried out. Others who kindly provided antibodies were Rainer Duden (rabbit anti+COP), Paul Luzio (anti-TGN 38), Frances Brodsky (anti-clathrin heavy chain), and Eric Berger (antigalactosyltransferase). We also thank Rick Klausner for the BFA analogs and for sending us preprints of unpublished work. T. E. K. thanks Brigitte Joggerst for excellent technical assistance. M. S. R. acknowledges support from the Wellcome Trust and the MRC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

1, 1991; revised

January

17. 1992

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