β-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo

Cell, Vol. 74, 71-62,

July 16, 1993. Copyright

0 1993 by Cell Press

P-COP Is Essential for Biosynthetic Membrane Transport from the Endoplasmic Reticulum to the Golgi Complex In Vivo Ft. Pepperkok,‘t J. Scheel,‘t H. Horstmann; H. P. Hauri,* G. Griffiths; and T. E. Kreis”t ‘European Molecular Biology Laboratory Meyerhofstrasse 1 D-6900 Heidelberg Federal Republic of Germany *Department of Pharmacology Biocenter Kiingeibergstrasse 70 CH-4065 Base1 Switzerland

Summary Microinjection of antibodies against a synthetic pep tide of a non-clathrin-coated vesicle-associated coat protein, p-COP, blocks transport of a temperaturesensitive vesicular stomatitis virus glycoprotein (ts045-G) to the cell surface. Transport is inhibited upon release of the viral glycoprotein from temperature blocks at 39.5% (endopiasmic reticulum [El?]) and 15% (intermediate compartment), but not at 20% (trans-Golgi network). Ts-045-G is arrested in tubular membrane structures containing ~53 at the interface of the ER and the Golgi stack. This is consistent with inhibition of acquisition of endoglycosidase H resistance of ts-045-G in Injected cells. Secretlon of endogenous proteins and maturation of cathepsin D are also inhibited. These data provide in vivo evidence that 9-COP has an Important function in biosynthetic membrane traffic in mammalian cells. Introduction Coated vesicles are involved in cytoplasmic membrane traffic. Clathrin-coated vesicles containing specific adaptor complexes, HA1 and HA2, mediate receptordependent biosynthetic transport from the trans-Golgi network (TGN) toward endosomes and endocytic transport from the plasma membrane toward early endosomes, respectively (Kornfeid and Mellman, 1969; Pearse and Robinson, 1990). The role of the coat protein (COP)coated vesicles in membrane transport is less well understood. In vitro, they have been implicated in signal-independent bulk flow transport through cisternae of the Golgi complex (Rothman and Orci, 1992); in addition, their immunolocalization to the cis-Golgi network (CGN) and the TGN may also suggest a role in membrane transport toward and from theGoigi complex(Dudenet al., 1991; Oprinset al., 1993). Although Golgi complex-derived clathrin- and COPcoated vesicles are morphologically and functionally different, the sequence homology of a non-clathrin-coated

tPresent UniversitB,

address: DBpartement de Biologic Cellulaire, 30. quai Ernest Ansermet, CH-1211 GenBve,

Sciences Ill, Switzerland.

vesicle-associated COP, p-COP, with 8-adaptin (Duden et al., 1991) and the similar molecular masses of the proteins in the two classes of coat complexes (Waters et al., 1991) indicate that their functions may be related. Treatment of cells with brefeldin A, a fungal metabolite that dramatically disorganizes endoceliuiar membranes, has led to further insights into the machinery regulating membrane transport (Kiausner et al., 1992). in these drugtreated cells, p-COP is rapidly removed from the Golgi complex (Donaldson et al., 1990; Oprins et al., 1993). Brefeldin A also leads to the rapid redistribution of y-adaptin (but not a-adaptin; Robinson and Kreis, 1992; Wong and Brodsky, 1992) a component of HAl, further indicating that the two different coat protein systems associated with membranes of the Golgi complex may have related functions. Binding of COPS to membranes depends on GTPbinding proteins (Melancon et al., 1967; Donaldson et al., 1991; Robinson and Kreis, 1992). ADP-ribosylation factor (Donaldson et al., 1992a) and Goigi membrane enzymes catalyzing exchange of nucieotides (Donaldson et al., 1992b; Helms and Rothman, 1992) have been implicated in the regulation of COP-membrane interactions. Upon longer incubation of ceils with brefeidin A, membranes of the Golgi complex fuse with membranes of the endopiasmic reticulum (ER) (Doms et al., 1969; Lippincott-Schwartz et al., 1969). Collectively, these observations led to the hypothesis that COPS play essential roles in regulating antero- or retrograde transport between these cytoplasmic compartments (Klausner et al., 1992; Kreis, 1992; Mellman and Simon% 1992). The development of cell-free transport systems and the isolation of yeast secretory mutants have opened the way for a dissection of the protein machinery involved in processes of membrane transport toward and through the Golgi complex and have allowed visualization of transport intermediates by electron microscopy (Novick et al., 1980; Orci et al., 1986, 1989; Malhotra et al., 1989; Kaiser and Schekman, 1990; Goda and Pfeffer, 1991; Plutner et al., 1991; Rexach and Schekman, 1991; Hosobuchi et al., 1992). Mutant cell lineswith impaired protein glycosylation and temperature-sensative mutant viral glycoproteins (e.g., ts-045-G) as models for maturation and biosynthetic transport of membrane proteins, have also been essential tools in the characterization of these processes in vitro and in vivo. Ts-045-G is blocked in the ER at nonpermissive temperature (39.5%); it accumulates in the intermediate compartment, the interface of the ER, and the ciscisternae of the Golgi stack (Schweizer et al., 1990; Lotti et al., 1992) at 15% and in the TGN at 20% (Griffiths and Simon% 1986). Shifting infected cells to permissive temperature (31 “C) induces normal transport of the viral glycoprotein from these compartments to the cell surface. These features of the transport pathway of ts-045-G facilitated the analysis of the biosynthetic membrane traffic in vivo. We have studied the role of B-COP, the best-characterized component of the COP complex so far, in biosyn-

Cell 72

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acids

8 110-12 110-5 EAGE El Fl 110-6 KESE Dl maD KLVE 110-7 110-8 LGDK

16-29 36-53 69-83 84-97 107-120 147-157 161-175 198-211 249-26 1 291-307 329-342 361-375 391-411 453-467 496-513 529-542 597-611 618-631 656-669 701-715 701-715 767-786 826-839 871-887 940-953

1. Characterization

of Antibodies

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One MAb (maD) and the 24 polyclonal antibodies against synthetic peptktes of b-COP were characterized by immunoblotting on rat liver and Vero cytosol, by indirect immunofluorescence on methanol-fixed Vero cells, by binding to native protein in streptolysin 0-permeabilized Vero ceils (cell models), and by microinjection into living Vero cells (for details see Experimental Procedures). The position of the peptides and a summary of their reaction in the various tests shown in (6) are summarized in (A). Antibodies against underlined peptides react only by immunoblotting with the rat protein, in open boxes by immunoblotting and in stippled boxes by immunofluorescence on Vero cells. Closed arrowheads indicate those antibodies that react with native protein in cell models, and an additional arrowhead indicates binding of microinjected antibodies to g-COP in vivo. Plus sign, positive reaction; minus sign, negative reaction; n. d., not determined; asterisk, positive only in 30%~50% of VW-infected cells.

thetic membrane traffic. A battery of epitope-specific antibodies against this COP was generated to test their effects on biosynthetic transport of ts-045-G upon microinjection into living cells. Transport and maturation of the viral membrane protein were used as indicators for the analysis of effects of injected antibodies in these cells using biochemical methods and immunolocalization on the light and electron microscope level. The effects of these microinjected antibodies on maturation and secretion of endogenous cellular proteins were also analyzed in vivo. The results argue strongly that P-COP plays an essential role in the biosynthetic pathway of mammalian cells. Results Effect of Microlnjected Antibodies against Synthetic Peptldes of jKOP on the Morphology of the Golgi Complex and Adjacent Compartments A library of epitope-specific antibodies was generated in rabbits against 24 synthetic peptides of f&COP. Affinity-

purified antibodies were characterized by immunoblotting, immunofluorescence microscopy, and microinjection into Vero cells (Figure 1). Of the 18 antibodies that reacted with rat B-COP by immunoblotting, 11 cross-reacted with the primate (Vero cell) protein. Four of the nine antibodies positive by immunofluorescence microscopy on Vero cells also labeled native P-COP in cell models, and two (antiEAGE and anti-l 1O-l 2) bound to the Golgi-associated protein when microinjected into living cells. No labeling of the region of the Golgi complex was detected when cells were microinjected with these antibodies preincubated with the corresponding peptides. Interestingly, microinjected polyclonal antibodies against peptides Dl and El, as well as the monoclonal antibody (MAb) against Dl (maD), gave a weak Golgi staining in 30%-50% of vesicular stomatitis virus (VW)-infected cells. The effects of microinjected anti-EAGE and anti-l lo-12 on organelles involved in biosynthetic membrane transport were analyzed at the light (Figure 2) and electron microscope level (see Figure 7). At 1.5-2 mglml (corre-

F$ZOP Is Essential

in Biosynthetic

Membrane

Traffic

Figure 2. Effect of Microinjected Anti-EAGE on the Distributiin of Protelns Associated with the Gofgi Complex and Adjacent Membranes Polyctonal rabbit antibodies (1.3 mglml) against the EAGE peptide of 3COP were microinjected into Vero c&e. Cetls were fixed 2 hr posttnjectton, and injected anti-EAGE was labeled with fluoreecein-conjugated secondary antibodies (a, c, e, and g). C&t were doublelabeled wtth maD (b), the 33K Gotgiiiated protein (d). p33 intermediate compartmentassociated protein (9, or -+daptin (h) and stained with rhodamine-conjugated secondary antibodies. Injected anti-EAGE completely co incided with mat3 (arrowbeads in [a] and [b]) and labehsd Gotgi membranes(d), but no overlap with clathrincoated veatctea was detected (compare large arrowbead wtth large arrow in [g] and [hD. Microtnjected antt-EAQE (intact antibodies or monovalent Fab fragments) induced protiferatton of tubular structures labeled with antibodies against p53 (arrows in [fJ). Bar, 20 Wm.

sponding to - 100 ug of antibodies per milliliter of cytoplasm), microinjected anti-EAGE (Figures 2a and 2b) and anti-l 1O-l 2 (data not shown) rapidly labeled f3COP associated with the Golgi complex, as detected with maD in the fixed cells. The organization of the Golgi complex, visualized with an antibody against a Golgi stack-associated 58K protein (Bloom and Brashear, 1989) or labeled with NED-ceramide metabolites (data not shown), and the distribution of TGN-derived clathrin-coated vesicles labeled with an antibody against y-adaptin (Ahle et al., 1988) appeared unaffected by the microinjected antibodies (Figures 2c, 2d, 29, and 2h). Microinjection of anti-EAGE had also no effect on the distribution of the ER marker protein PDI (data not shown). Of the two antibodies that labeled P-COP in injected cells, anti-EAGE, however, rapidly (within - 15 min) led to the reproducible rearrangement and apparent tubularization of membranes labeled with anti-p53 (Figures 26 and 29. Microinjected anti-l 1 O-l 2, on the other hand, had no such effect (not shown). Upon

longer incubation (l-2 days) of cells microinjected with anti-EAGE, the Golgi complex contracted, the cells appeared smaller in size, and they finally detached from the coverslips (data not shown). Interestingly, “clumps” with labeled r-adaptin overlapped with P-COP-positive structures in these cells, and only a few vesicular profiles positive for either of the two COPS remained in the cytoplasm. Vero cells microinjected either with mono- or divalent anti-EAGE or anti-l 1 O-12 were analyzed 2 hr postinjection by electron microscopy following embedding in Epon. Microinjected cells were identified in the thin sections by including bovine serum albumin coupled to 5 nm gold particles in the injection solution. In cells microinjected with anti-EAGE (intact or Fab fragments at 1.8 mglml), the structure of the Golgi stacks remained intact; however, an extensive proliferation of tubular structures on one side of the Golgi stack was observed (see small arrowheads in Figures 7b and 7~). This is most likely the cis side of the Golgi complex since continuity of tubular extensions

Cdl 74

Figure 3. Microinjected Anti-EAGE Interferes with Appearance of T&45-G on the Cell Surface Vero cells were microinjected at 39.5OC with anti-EAGE (a, c, and e) or anti-11912 (b, d, and 9 2 hr after infection with W-G45 VSV. Cells were shifted 30 min after injectiin to permissive temperature (31 “C) in the presence of cycloheximide and fixed 45 min later to covisualize ts-045-G on the cell surface (c and d) and injected antibodies in the cytoplasm (e and 9 as described in Experimental Procedures. (a) and (b) show the corresponding cells by phasecontrast microscopy. No viral glycopmtein is detected with MAb VG on the surface of cells injected with anti-EAGE (cand e), whereas normal surface staining of teO45G can be obsewed in cells injected with anti-l IO-12 and neighboring noninjected cells. Bar. 29 nm.

with membranes of the rough ER could be detected (see large arrowhead in Figure 7b) and since the tubules appeared associated with fenestrated membranes on the cis side of the Golgi complex (cf. Rambourg and Clermont, 1990). Also striking was the appearance of electron-dense spherical structures continuous with the proliferating tubules (see Figures 7c and 7d; see also Oprins et al., 1993; Hendricks et al., 1992). These tubular profiles and spherical structures were absent in control cells (noninjected or injected with anti-l 10-12). Effect of Microinjected Anti-j&COP Antibodies on Transport of Ts-045-G to the Cell Surface Ts-045 WY/-infected Vero cells were injected 2 hr postinfection at nonpermissive temperature (39.5%) with different antibodies against P-COP peptides (anti-EAGE and anti-l 10-12, 1.8 mglml; anti-Al, 2.7 mglml; anti-D1 and anti-El, 3 mglml). Cells were shifted to permissive temperature (31 ‘X) in the presence of cycloheximide 30 min after injection, and the appearance of ts-045-G at the cell surface was analyzed before permeabilization with MAb VG

reacting with the extracellular domain of the viral glycoprotein (Figures 3-5). No ts-045-G could be detected on the plasma membrane of cells microinjected with divalent or monovalent anti-EAGE (Figures 3a, 3c, and 3e), whereas neighboring noninjected cells or cells injected with divalent anti-l lo-12 (Figures 3b, 3d, and 39 exhibited normal levels of ts-045-G on the cell surface. The rate of appearance (Figure 4) and the amounts (Figure 5) of ts-045-G on the cell surface were determined by immunofluorescence and quantitative image analysis in cells injected with several different antibodies against f&COP peptides. Fab fragments and intact anti-EAGE blocked transport of ts-045-G to the cell surface, whereas in control cells injected with anti-l 1 O-l 2 or anti-EAGE preincubated with the EAGE peptide (loo- to 500-fold molar excess), no significant inhibition in the rate of transport (compared with uninjected cells) could be detected (see Figure 4). Microinjected EAGE peptide alone also had no inhibitory effect (data not shown). The cells injected with antibodies against the peptides 110-12, Al, Dl, or El showed normal ts-045-G surface staining (Figure 5). In

f35COP Is Essential

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in Biosynthetic

100 80 80 40

Membrane

Traffic

/. -.-.-.-.___.___ - *-._.q-..--o--.

uninpcted EKE F&AGE EAGE+peptde 110-12

20 0

Figure 4. Rate of Appearance jetted Cells

of Ts-045-G

on the Surface

of Microin-

Virus-infected Vero cells were microinjected with anti-EAGE (1.5 mgl ml), anti-EAGE Fab fragments (2 mg/ml). anti-EAGE preincubated with EAGE peptide (5 mglml), and anti-l 10-12 (1.8 mglml), as described in Figure 3. At different time points after shifting to permissive temperature, cells were fixed and double-labeled for injected antibodies and for viral glycoprotein on the surface. Fluorescence of k-045-G on the cell surface was quantified (for details see Experimental Procedures). Each data point represents the mean of two independent experiments with at least 50 cells analyzed. Maximal deviation from the mean was always below 15%. The values are normalized to the highest value obtained for surface fluorescence in noninjected cells (100%).

contrast, less than 15% of the amount of surface staining obtained in control ceils was measured in cells microinjetted with anti-EAGE or its Fab fragments for up to 3 hr upon shift to permissive temperature (Figures 4 and 5). Mlcroinjected Anti-EAGE Blocks Biosynthetic Transport of Te-045-G at the Interface of the ER and the Golgi Stack The cytoplasmic compartment where microinjected antiEAGE blocks transport of t.s-045G was next analyzed by temperature shift experiments, quantitative immunofluorescence, immunoelectron microscopy, and biochemical methods. VSV-infected cells were microinjected at 39.5OC, 15OC, or 20°C with t&45-G arrested in the ER, in the ER to Golgi intermediate compartment, or in the TGN, respectively. Cells were shifted 36 min postinjection for 66 min to the permissive temperature, and the amount of ts045-G at the cell surface was quantified by immunofluorescence and image analysis (Figure 5). Microinjection of anti-EAGE at 39X and 15°C resulted in the arrest of transport of ts-O45-G to the cell surface, but no significant inhibition was observed when cells were injected after accumulation of ts-045-G in the TGN at 2O’X.Z (no ts-045-G could be detected on the surface of cells kept at 20°C; data not shown). We concluded therefore that transport to the cell surface of ts-045-G is blocked by injected antiEAGE before it reaches the TGN. The structures where ts-O45-G accumulates in injected cells were localized in a first attempt by triple immunofluorescence labeling: infected cells were injected with antiEAGE at 39.5OC, and ts-045-G transport was released by shifting cells 30 min postinjection to 31 OC for 60 min (Figure 6). Fixed cells were labeled with specific antibodies for ts-045-G (Figure 6a), p53 (Figure 6b), and microinjected anti-EAGE (Figure 6~). Accumulation of tubular structures

was observed in injected cells (Figures 6a and 6b; see also Figure 29, and significant colocalization of ~53, ts-045-G, and microinjected anti-EAGE was detected. The tubular structures, clearly positive for p53 and ts-045-G, appeared, however, only weakly labeled with injected antiEAGE antibodies. In neighboring uninjected cells, ts045-G had reached the Golgi complex (Figure 6a, left cell), and the bulk of p53 colocalized with this region of the Golgi complex. Extensive tubular structures could also be detected when cells were injected with anti-EAGE at 15OC after accumulation of ts-045-G for 2 hr in the intermediate compartment and then shifted for 60 min to permissive temperature (Figures 6d and Se). Again, the tubules containing the viral glycoprotein (arrows in Figure 6d) labeled only weakly with injected anti-EAGE. Remarkably, however, patches enriched in ts-045-G and P-COP were often found at ends of these tubules (arrowheads in Figures 6d and Se). We concluded that injected anti-EAGE blocks ts-045-G in tubular membranes containing ~53. To test further whether ts-045-G reaches the stacked Golgi cisternae upon microinjection of anti-EAGE antibodies, cells were freeze-substituted in Lowicryl HM20, and sections were labeled with antibodies against the viral glycoprotein (Figure 7e). We could not detect significant labeling of ts-045-G on the surface of injected cells, and no profiles of budding virus particles were observed (Figure 7e). In only a few cells could labeling of ts-045-G be detected in proximity to the Golgi stack, and it was always confined to one side of this organelle. Most of the goldlabeled ts-O45-G was distributed over the cytoplasm, but owing to the poor visualization of membranes in Lowicryl sections, it wasdifficult to recognize the labeled structures (Figure 7e). The cells injected with control antibodies (anti11 O-l 2) and noninjected cells had significant ts-045-G labeling both on the plasma mem brane, where budding virus particles could often be seen (data not shown), and on the Golgi stack (insert in Figure 78). Collectively, our electron microscopy data suggest that an extensive proliferation of pre-Golgi smooth membranes occurs in anti-EAGEinjected cells. Further, injected antibodies against this peptide arrest ts-045-G at a stage before it is transported into the Golgi stack. In addition to the immunolocalization of arrested ts045-G in injected cells, we also used endoglycosidase H (endo H) resistance as an indicator for whether or not the viral glycoprotein had reached the stacks of the Golgi cisternae (Figure 6). Approximately 596 cells that had been plated in a drop on a glass coverslip were infected with ts-045 VSV for 30 min at 39.5OC and then microinjected at 39.5”C using an automated microinjection system (Pepperkok et al., 1968). After microinjection, cells were incubated for a further 90 min and subsequently pulse-labeled with [35S]methionine for 15 min at 39.5OC. The temperature was then shifted to 31 “C for 90 min before ts-045-G was immunoprecipitated and analyzed for its sensitivity to endo H. At the end of the pulse, t&45-G was completely sensitive to the glycosidase; after 96 min of chase, only 14% of the ts-045-G in noninjected cells and 17% in control cells injected with anti-Al remained sensitive to endo H (Figure 6). In cells injected with anti-EAGE, 43% of ts-

Cell 76

T

I3 • a

Al Dl El

Figure 5. Anti-EAGE Blocks Transport of Ts045-G to the Cell Surface between the ER and the TGN

Vero cells were infected with ts-045 VSV, and the viral glycoprotein was accumulated for 2 hr 110-12 at 39.5OC in the ER, at 1WC in the intermediate 60 EPGE compartment, and at 20% in the TGN in the FabEAGE presence of cycloheximide (for details see Experimental Procedures). Cells were then microinjected at the respective temperatures in the presence of cycloheximide with different antibodies against B-COP (anti-EAGE and Fab fragments anti-EAGE and anti-l 10-12, 1.6-2 20 mglml; anti-Al, 2.7 mg/ml; anti-D1 and anti-El, 3 mg/ml) and, 30 min postinjection, shifted for 60 min to permissive temperature. The amount of viral glycoprotein that had reached the cell surface was quantified as described in detail in Experimental Procedures. Each data point is normalized to the value for surface staining obtained for noninjected cells from the corresponding coverslip.

045-G remained sensitive to endo H, suggesting that injected anti-EAGE significantly inhibited its transport into the Golgi stack. In parallel control experiments, it was established by immunofluorescence for ts-045-G and injected anti-EAGE that more than 85% of the infected cells on the coverslip had been injected with antibodies. Possible explanations for the incomplete block of arrival of ts045-G in the Golgi cisternae may be that the 15% noninjetted cells contributed significantly to the fraction of endo H-resistant ts-045-G or that the injected antibody-induced effect is leaky with respect to transport into (but not out 09 the Golgi complex. Effect of Microinjected Anti-EAGE on Biosynthetic Transport of Endogenous Proteins in Noninfected Cells To test the effect of microinjected antibodies on overall protein secretion, the medium of -500 metabolically labeled, microinjected cells was analyzed. Proteins in the medium (and in the cell lysates) were precipitated with trichloroacetic acid and quantified by precipitated radioactivity. Microinjection of anti-Al did not significantly decrease secretion (108 f 10% of the value of noninjected control cells); microinjection of anti-EAGE, however, inhibited secretion of trichloroacetic acid-precipitable protein more than 3-fold (31 f 10%). Analysis of the precipitated material by SDS-polyacrylamide gel electrophoresis confirmed this result and, furthermore, revealed that secretion was only quantitatively affected, since no major differences in the secreted protein patterns could be detected when secreted proteins were compared in control, anti-Al injected, and anti-EAGE-injected cells (data not shown). We concluded that microinjection of anti-EAGE inhibits transport of secretory proteins to the cell surface. Maturation of cathepsin D, a lysosomal hydrolase, was used as a model for studying the effect of injected antiEAGE on the transport of a defined endogenous protein in noninfected cells. Cathepsin D is proteolytically processed during its transport from the ER through the Golgi complex to endosomes and lysosomes (Gieselmann et al., 1985). It is synthesized as an -45 kd precursor and processed to an - 33 kd form in Vero cells (Figure 9). Since only the

45 kd immature form is missorted in the TGN and secreted into the medium (Figure 9) it can be assumed that proteolytic processing to the 33 kd form occurs after the Golgi complex, probably in endosomes. We observed that mi-

Figure 6. Ts-046-G Accumulates in Tubular p53 in Cells Injected with Anti-EAGE

Structures

Containing

T&45 VSV-infected Vero cells were injected with anti-EAGE at 39.5W (a-c) or 15% (d and e) and shifted to 31% as described in the legend to Figure 5. After 60 min at permissive temperature, cells were fixed, and triple immunofluorescence for the viral glycoprotein (a), p53(b), and injected anti-EAGE (c)or double immunofluorescence for ts-045-G (d) and injected anti-EAGE (e) were performed as described in Experimental Procedures. Arrows indicate tubular elements positive fort&Xi-G and ~53, but not p-COP, with small arrowheads showing patches containing t&46-G and B-COP at the ends of tubular structures; the larger arrowhead depicts membranes labeled for all three antigens. Ts-045-G reached the Golgi complex under these conditions in noninjected cells (cell to the left in [a]). Bar, 20 pm.

j3COP 77

Is Essential

in Biosynthetic

Figure

7. Electron

Microscopic

Membrane

Analysis

Traffic

of Cells

Microinjected

with Anti-EAGE

Electron micrographs of Epon sections of Vero cells injected with either control antibodies (anti-l 10-12) (a) or anti-EAGE (b and c) are shown. Cells were fixed 2 hr after microinjection. (b and c) Tubular elements (small arrowheads) accumulate on one side of the Golgi stack (G). The large arrowhead indicates continuity between the rough ER and the tubular elements. In (c)the fenestrated appearance of cis-Golgi elements is apparent, and an electron-dense area (asterisk) is shown in close contiguity with the tubular elements. Both structures were observed only in cells microinjected with anti-EAGE. In Epon sections of injected, infected cells, strong labeling is seen on the periphery of the electron-dense structures (d). (e) Vero cells were infected with ts-045 VW, microinjected with anti-EAGE or control anti-l lo-12 (insert) and bovine serum albumin coupled to 5 nm of gold particles, and shifted for 1 hr to permissive temperature as described in the legend to Figure 3. Lowicryl sections of freeze-substituted cells were labeled with anti-VSV G spike followed by protein A-gold. In cells microinjected with control antibodies, strong labeling of ts-045-G on the Golgi stack is seen (insert), but only rarely in the immediate vicinity of Golgi cisternae in cells injected with anti-EAGE (e). The membrane in anti-EAGE-injected cells shows no viral glycoproteins, and no budding virus particles are seen (e). Arrows in (e) indicate scattered gold particles on illdefined parts of the cytoplasm labeling ts-045-G in anti-EAGE-injected cells. G indicates the Golgi stack; N, a nucleus; and R, the rough ER. Bars in (a-d), 0.2 pm; bar in (e), 0.1 urn.

Cell 70

Ab

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Figure 8. Acquisition of Endo H Resistance in Cells Injected with Anti-EAGE

+

of Ts-045-G

Is Inhibited

Approximately 500 Vero cells grown on small coverslips were infected with ts-045 VW. Cells were microinjected at 39.5W with anti-EAGE or anti-Al between 30 and 50 min postinfection. After another 90 min at nonpermissive temperature, noninjected cells(n) and cells injected with anti-EAGE (EAGE) or anti-Al (Al) were metabolically labeled with [35S]methionine. Ts-045-G was immunoprecipitated either directly after the [35S]methionine pulse (0) or after a further 90 min chase at 31 OC (90) and was divided into two equal aliquots, of which one (plus) was digested with endo H. Ts-045-G in noninjected cells was fully sensitive to endo H at nonpermissive temperature; 90 min after shifting to 31 OC, only 14%-170/o of the viral glycoprotein remained sensitive to endo H in noninjected or anti-Al-injected cells. In cells injected with anti-EAGE. 43% of the ts-045-G remained sensitive to endo H.

croinjected anti-EAGE inhibits the processing of cathepsin D: only 7% of cathepsin D is processed to the 33 kd form after a 90 min chase in injected cells, whereas 36% or 33% of cathepsin D is processed in noninjected or anti-Al injected control cells, respectively. Thus, microinjection of anti-EAGE antibodies also inhibits the intracellular transport of a defined endogenous protein, cathepsin D. Discussion The temperature-sensitive viral glycoprotein, ts-045-G, and microinjection of epitope-specific antibodies have been used to analyze the role of P-COP, a non-clathrincoated vesicle-associated COP, in biosynthetic membrane transport from the ER through the Golgi complex to the cell surface. Microinjected monovalent antibodies against B-COP (anti-EAGE at <2 mglml, equivalent to - 100 uglml in the cytoplasm of injected cells) block transport of ts-045-G to the cell surface before it enters the Golgi stack. Furthermore, transport and maturation of an endogenous protein, cathepsin D, as well as protein secretion, are inhibited. These results strongly suggest in vivo that p-COP is required for biosynthetic membrane transport at the ER to Golgi interface in mammalian cells. The recent finding that the 105 kd gene product of S/X27, an essential gene for protein transport from the ER to the Golgi in Saccharomyces cerevisiae, is a subunit of coatomer (Hosobuchi et al., 1992; see also Stenbeck et al., 1992) strongly supports this conclusion. We therefore consider it unlikely that an indirect antibody effect or an antibody-induced P-COP-mediated transport inhibition was measured in our experiments. Thus, COPS are required for the regulation of early steps in membrane traffic in eukaryotic cells as different as yeast and primates. The morphological analysis of the cells injected with the anti-EAGE antibodies revealed an extended tubular network of membranes, containing the intermediate compartment marker protein ~53, that forms in both uninfected

and infected cells. The simplest explanation for this observation is that membrane synthesis continues while budding of COP-coated carrier vesicles and transport is arrested, resulting in the accumulation of membranes in tubular structures adjacent to the site of the block. It also renders the possibility unlikely that these COPS regulate anterograde membranetraffic by inhibiting budding. Since no accumulation of vesicular transport intermediates could be found by immunofluorescence or electron microscopy of microinjected cells, COPS appear to support the budding process. In addition, no significant effect of the injected antibodies was detected within several hours on the morphology of the stacked Golgi cisternae, indicating that blocking forward membrane traffic between the ER and the cis-Golgi cisternae does not itself lead to the disappearance of the Golgi complex due to membrane depletion. Further information will be obtained by direct visualization of these processes in living cells. It is conceivable that intra-Golgi transport may also be inhibited by anti-COP antibodies; COP-coated vesicles have been implicated in bulk flow transfer of material through the stacked Golgi cisternae (Rothman and Orci, 1992). The effect of microinjected antibodies on transport of ts-045-G through the stack of Golgi cisternae could, however, not be assayed in vivo, since no reversible block has yet been found that leads to the accumulation of viral glycoprotein in cisternae of the Golgi stack. Clearly, transport of ts-045-G from the TGN to the cell surface is not affected by injected antibodies, although some labeling of the TGN with antibodies against P-COP has been observed in Vero cells (Duden et al., 1991; G. G. and T. E. K., unpublished data) and in rat exocrine pancreas cells (Oprins et al., 1993). Several explanations for these observations can be discussed. First, although p-COP can be localized on membranes of the TGN, COPS may be inactive at this compartment. It is also possible that anti-EAGE cannot bind to TGN-associated P-COP in vivo because this site is blocked owing to its interaction

I

chase

cell lysates -

0

1

2

seer.

3

Figure 9. Transport of Cathepsin with Anti-EAGE Antibodies

Ab.

3

D Is Inhibited

n

Al

1.5

1.5

EAGE

15

in Cells Microinjected

Vero ceils were metabolically labeled with [36S]methionine for 15 min and subsequently chased for up to 3 hr. Cathepsin D was immunoprecipitated from cell lysates and culture supernatants after the indicated hours of chase and was analyzed by SDS-polyacrylamide gel electrophoresis. The positions of the precursor (open rhombus) and the processed form (closed rhombus) of cathepsin D are indicated. Only the precursor form is secreted into the medium (seer). Microinjection of anti-EAGE (see the legend to Figure 9) inhibits the processing of cathepsin D to the mature form, whereas injection of control antibodies has no effect. Molecular mass standards are in kilodaltons.

P-COP 79

Is Essential

in Biosynthetic

Membrane

Traffic

with another TGN-specificcomponent. In addition, a family of p-COP proteins may exist, each member of this family involved in one defined step of membrane transport. The precise molecular mechanism by which microinjetted antibodies against the EAGE epitope of B-COP interfere with the function of the COPS is not known. Clearly, monovalent antibodies have the same inhibitory effect as the divalent antibodies, ruling out the possibility that COPS are inactivated simply by aggregation. Furthermore, P-COP is not released from membranes by the injected antibodies, in contrast with the rapid dissociation of COPS from Golgi membranes by energy depletion or brefeldin A (Donaldson et al., 1990). Obviously, several possible mechanisms can be postulated to explain the inhibition of the function of P-COP by injected antibodies. We consider the following two the most likely. First, injected antibodies bound to P-COP may sterically interfere with its interaction with other factors essential to the proper functioning of the coated vesicle machinery (i.e., binding to a receptor on a target membrane, assembly of a functional coat complex, interaction with a soluble factor that triggers the coatmediated vesicle budding). Alternatively, injected antiEAGE antibodies interfere with conformational changes of P-COP that are necessary for its functional activity. Two dozen peptide antibodies recognizing epitopes distributed over the entire length of p-COP have been characterized and tested. Four recognize the native protein, two (anti-EAGE and anti-l 10-12) bind to Golgi-associated B-COP in living cells, yet only one (anti-EAGE) interferes with biosynthetic membrane transport in vivo. This result strongly suggests that the larger part of Golgi-bound p-COP is buried within a complex in vivo. Interestingly, the EAGE epitope is located in the center of the f%COP sequence (amino acids 496-513). This domain is located toward the C-terminal end of the region of homology of P-COP with 9-adaptin, close (- 50 amino acids N-terminal) to the proline-rich hinge region of f3-adaptin (Duden et al., 1991; Matsui and Kirchhausen, 1990; Pearse and Robinson, 1990). It is conceivable that an antibody bound to f3-COP in this region may alter the conformational properties of P-COP. Yet, since this region is accessible only to the injected antibodies against the EAGE and 110-l 2 peptides, this is also the likeliest domain where p-COP may interact with other (non-coatomer) factors. p-COP is, in fact, associated with a high molecular weight complex, inactivation of which with anti-P-COP peptide antibodies interferes with export from the ER in vitro (Peter et al., submitted). Saturation of the biosynthetic transport machinery with viral glycoproteins may deplete such factors from the cytoplasm, resulting in the exposure of normally cryptic epitopes on 9COP (the Dl and El epitopes are only accessible in vivo to injected antibodies in virusinfected cells; Figure 1). Microinjection of epitope-specific antibodies in combination with biochemical and structural analysis has proven a powerful approach for characterizing the essential role of a component of the molecular machinery involved in cytoplasmic membrane traffic. Further experiments in vivo and in vitro on the molecular interactions of B-COP with

other components in the COP complex are required to clarify the precise mechanism of COP function and may lead to the identification of additional factors that interact with the COP complex. Experimental

Proceduras

AntibodIes Antibodies against synthetic peptides of B-COP have been prepared and affinity purified as described (Duden et al., 1991; Kreis, 1966). A murine MAb (maD) against peptide Dl was prepared using a method described previously (Kreis, 1966). Murine MAbs against p53 (Schweizer et al., 1966) 56K (Bloom and Brashear, 1969), PDI (ID3, culture supernatant jVaux et al., 19901). v-adaptin (Ahle et al., 1966) VSV glycoprotein (P5D4 [Kreis, 1966); MAb VG [K. Simons, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany]), affinity-purified rabbit antibodies against VSV glycoprotein (aP4 [Kreis, 19661; anti-VSV G spike [Griffiths et al., 19651) and rabbit polyclonal antiserum against cathepsin D (from Drs. K. F. Johnson and S. Kornfeld, Washington University, St. Louis, Missouri) were also used. Fab fragments of antibodies were prepared according to the instructions of the manufacturer using papain immobilized on beads (Pierce Chemical Company, Rockford, Illinois). Cell Culture and Vlrus InfactIons Vero cells (African green monkey kidney cells, ATCC CCL 61) were maintained and infected with ts-045 VSV (Indiana serotype) as described earlier (Kreis and Lodish, 1966).

Mlcrolnjactlon Capillary microinjection of antibodies into cells was performed with an automated microinjection system (Zeiss AIS; Ansorge and Pepperkok. 1966). All microinjection experiments were carried out in 3 cm petri dishes containing 4 ml of Hanks’ medium with 5% fetal calf serum (FCS). The microscope stage was temperature controlled as described (Hung Leo et al., 1992). For labeling microinjected cells with [?Z]methionine and biochemical analyses, 500-1000 cells in culture medium were plated in droplets of 5 nl onto small glass coverslips and allowed to attach for 12 hr; 6 ml of medium was then added, and the cells were cultured for a further 36 hr before use.

Immunotluorescence

and Immunoalectron

Microscopy

Fixation using methanol or paraformaldehyde(for cell surface labeling) has been described (Kreis, 1966). For staining of ts-045-G at the surface, injected, paraformaldehyde-fixed cells were incubated for 15 min with MAb VG specific for a luminal epitope of the viral glycoprotein, followed after washing with phosphate-buffered saline (PBS) by incubation for 20 min with rhodamine anti-mouse antibody. Unbound secondary antibodies were washed away with PBS, cells were permeabilized, and injected antibodies were labeled with fluorescein anti-rabbit antibody as described (Kreis, 1966). For the simultaneous detection of injected antibodies (~53 and ts-045-G) methanol-fixed cells were incubated with biotin-conjugated anti-rabbit antibody and the MAb against p53 for 30 min, followed by incubation for 30 min with cascade blue-conjugated streptavidin (Molecular Probes Incorporated, Eugene, Oregon) and fluorescein anti-mouse antibody. Unoccupied binding sitesof the anti-mouse antibodies were blocked with purified mouse immunoglobulin G (final concentration, 1 mg/ml; Sigma Chemical Company, St. Louis, Missouri) for 20 min. Cellular ts-045-G was then labeled directly for 30 min with rhodamine-conjugated MAb P5D4 (Kreis, 1966). Cells were permeabilized with streptolysin 0 to analyze binding of antibodies to native protein in cell models. Vero cells were incubated for 15 min at 20% with lysis buffer (100 mM K-PIPES, 5 mM EGTA. 5 mM MgCI,, 1 mM dithiothreitol, 0.5 uM taxol [pli 6.61) containing 4 IUlml equivalents of streptolysin 0 (precipitated with 75% ammonium sulfate and dialyzed against 100 mM K-PIPES, 5 mM EGTA, 5 mM magnesium acetate [pH 6.61; Wellcome Biotechnology, Beckenham, England), 20 pM GTPrS. and rhodamine-labeled Fab fragments of a MAb against tyrosinated a-tubulin (1 A2; Kreis, 1967) to identify permeabilized cells and the relevant antibodies against COP peptides. Cells

Cell 80

were subsequently rinsed twice for 1 min at 20°C with lysis buffer to remove unbound antibodies and subsequently fixed for 4 min in methanol at -20°C. The distribution of antibodies against COP peptides were visualized with fluorescein-labeled secondary antibodies. Coverslips were scratched with a compass to locate microinjected cells for electron microscopic analyses. For fixation, cells were briefly rinsed in PBS and then placed in 8% paraformaldehyde in 250 mM HEPES(pH 7.4)for4 hr followed by2.1 M sucrose in PBS(lOmin)prior to freezing in liquid nitrogen. The latter method and the subsequent processing for freeze substitution in Lowicryl HM20 or Epon using the Reichert CS Auto was done as described by van Genderen et al. (1991). After polymerization of a thin (- 1 mm) layer of plastic onto the coverslip, the glass was dissolved in hydrofluoric acid. Subsequently, the thin piece of plastic containing the cell monolayer was mounted on a gelatin capsule containing unpolymerized Epon, and the sandwich was polymerized. The cell monolayer exposed on the surface of this block was then sectioned on a Reichert OM-2 microtome. For immunogold labeling, sections were placed on carboncoated Formvar grids and labeled. Nonspecific binding sites were blocked by incubating the sections in 10% FCS for 10 min, were rinsed four times for 10 min with PBS, and were incubated for 2.5 hr in PBS with 5% FCS containing antibodies. The grids were then incubated for another 2.5 hr in a solution of PBS containing 5% FCS and protein A-gold (9 nm). Sections were stained for 5 min in a I:1 mixture of saturated uranyl acetate and ethanol and 30 set on lead citrate. For preembedding labeling and embedding, cells were fixed in 1% glutaraldehyde in 0.2 M cacodylate with 0.05% saponin for 5 min and rinsed in PBS containing 20 mM glycin and 0.05% saponin for 30 min. The mouse MAb against the cytoplasmic domain of VSV glycoprotein (PSD4), diluted in PBS with 5% FCS, was added to the cells and incubated for 2.5 hr. After briefly rinsing the cells in PBS, goat antimouse antibodies conjugated with 9 nm of gold were added for another 2.5 hr. The cells were then fixed in 1% glutaraldehyde in 0.2 M cacodylate (pH 7.4) for 1 hr. Postfixation was done in 2% osmium tetroxide containing 1.5% potassium ferricyanide in water for 1 hr at room temperature. The cells were washed in water four times for 5 min, stained with 0.5% uranyl acetate for 30 min, and washed in water another four times. The preparation was dehydrated in ethanol and embedded in Epon. Fluorescence Microscopy and Quantltation of Cell Surface Fluorescence Microscopy and photography was performed on a Zeiss inverted fluerescence light microscope (Axioverl IO) equipped with a cooled CCD camera (1317 x 1035 pixels; Photometrics CH260) controlled by a Sun Microsystems workstation (IPX). Images taken with the CCD camera were further processed with the software package Khoros (Rasure et al., 1990) before printing on KodakTMAX IOOfilm, using aslidewriter (Type Honeywell, AGFA). For quantitation of t&45-G, surface-staining images of labeled cells were digitized using a SIT camera(Hamamatsu) coupled to a DVS image enhancement system (Hamamatsu). Background fluorescence was measured on cells stained only with rhodamine-conjugated secondary antibody and subtracted from images of labeled cells using the DVS system. For all these experiments, the same settings of the SIT camera and DVS system were used. Quantitation of surface fluorescence on digitized and background fluorescence-corrected images was performed with a Zeiss IBAS image analysis system. For this purpose, the periphery of cells to be analyzed was manually defined using the phase-contrast images of the cells, and the mean surface fluorescenceintensity(I,~)and thearea(A)of thecells weredetermined by the IBAS system. The integrated optical density (IOD) of the surface staining, which is proportional to the total amount of fluorescent molecules on the cell surface, was then determined by the formula IOD = A x I__ Metabolic Labeling of MIcroinjected Cells, Immunopreclpltation, and Endo H Treatment of Ts-046-G For virus experiments, infected cells were labeled and kept at 39.5’C until the start of the chase, whereas uninfected cells were labeled and maintained at 37OC for analysis of transport of cathepsin D or protein secretion. Microinjection of ts-045 VSV-infected cells was started 30-

60 min after shift to 39.5OC (1 hr after adsorption), and the cells were then allowed to recover for 90 min at 39.5OC. They were then incubated in methionine-free culture medium and, after 15 min, labeled with 2 mCi/ml of L-[3SS]methionine in methionine-free culture medium for 15 min at 37’C (uninfected cells) or 39.5OC (infected cells). Cells were washed afterward three times with chase medium (complete medium containing 5 mM methionine) and chased at 37OC (uninfected cells) or at 31°C in the presence of IO ug/ml cycloheximide (infected cells) for 90 min. At the end of the chase, culture supernatants were collected carefully, and cells were washed three times with chase medium and lysed in TNX (150 mM NaCI, 1 mM EDTA, 1% Triton X-100, 50 mM Tris-HCI [pH 7.41, supplemented with protease inhibitors [Scheel et al., 19901 and 1 mg/ml Vero cell extract that had been immunodepleted of cathepsin D). The media were supplemented with protease inhibitors and 0.5 mg/ml high speed supernatant from turkey liver as a carrier. Media and lysates were cleared by centrifugation for 10 min at 13,000 x g at 4OC. Ts-045-G was immunoprecipitated from cell lysates with aP4 and cathepsin D with a specific rabbit antiserum. Immunoprecipitation, digestion of immunoprecipitated protein with endo H, SDS-polyacrylamide gel electrophoresis, and fluorography were performed as described (Kreis and Lodish, 1986) except that Entensify (DuPont) was used as a fluorophor. Fluorographs were quantitated with an LKB Laser densitometer (Type Ultroscan XL, LKB Products, Bromma, Sweden) as described (Rosa et al., 1989). Proteins were precipitated from cell lysates or culture media by addition of 1 vol of 20% trichloroacetic acid in 50% acetone and incubation on ice for 3 hr. The precipitate was collected by centrifugation, washed with 50% acetone, dissolved in sample buffer, neutralized with ammonia vapor, and analyzed by liquid scintillation counting. The time course of maturation of cathepsin D was analyzed as described previously (Scheel et al., 1990). Acknowledgments We thank 8. Joggerst-Thomalla and A. Sawyer for excellent technical assistance. Generous gifts of antibodies from Drs. G. Bloom, R. Duden, S. Fuller, K. F. Johnson, S. Kornfeld, K. Simons, and E. Ungewickell are acknowledged. Taxol was obtained from Dr. N. Lomax (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute). We also would like to thank W. Ansorge for providing the microinjection and image-processing facilities and S. Herr for his help with the Khoros software package. We appreciated the helpful comments of W. Balch on the manuscript. R. P. was in part supported by a long-term fellowship from the European Molecular Biology Organization. Received

January

12, 1993; revised

April 26, 1993.

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