Cargo Can Modulate COPII Vesicle Formation from the Endoplasmic Reticulum*

Meir AridorDagger , Sergei I. Bannykh§, Tony Rowe, and William E. Balchparallel

From the Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
Top
Abstract
Introduction
References

The COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection. We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding. The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of alpha 1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58 in vitro. In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro. Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components. These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization. The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER.

    INTRODUCTION
Top
Abstract
Introduction
References

Newly synthesized (biosynthetic) cargo translocated into the endoplasmic reticulum (ER)1 is exported to downstream compartments by COPII vesicle carriers (reviewed in Refs. 1 and 2). Incorporation into vesicles is dependent on protein folding, a process that is stringently monitored by the ER (3). During export, biosynthetic cargo is sorted from resident ER proteins and selected for incorporation into vesicles by interacting with the Sar1 and Sec23/24 components of the COPII coat prior to vesicle formation (4, 5). Subsequently, the selected cargo incorporated into these pre-budding complexes is packaged into vesicles upon addition of the Sec13/31 complex. Because both membrane-associated proteins and soluble cargo proteins become physically associated with a pre-budding complex prior to export, it becomes important to determine whether biosynthetic cargo or cargo receptors can regulate vesicle coat assembly (4, 5), thereby linking cargo sorting to vesicle formation from the ER.

A role for cargo in directing vesicle formation was previously observed in both the late secretory pathway and the endocytic pathway (reviewed in Ref. 6). The transferrin receptor contains sorting determinants that are capable of interacting with the AP-2 complex that mediates clathrin-dependent endocytosis from the plasma membrane (reviewed in Ref. 7). Overexpression of transferrin leads to increased AP-2/clathrin coat recruitment to the plasma membrane and clathrin-coated endocytic vesicle formation (8, 9). Cargo proteins and cargo receptors in the trans-Golgi network (TGN), including mannose 6-phosphate receptors and the major histocompatibility class II protein complex, contain sorting determinants that are capable of binding to the AP-1 adaptor complex (7). The AP-1 complex mediates clathrin-dependent export of these proteins from the TGN (10-12). Overexpression or elimination of these cargo proteins affects both AP-1 coat recruitment and clathrin-coated vesicle formation.

To determine whether cargo exported from the ER is capable of modulating COPII vesicle formation, we have taken advantage of vesicular stomatitis virus (VSV)-infected cells to control biosynthetic cargo availability. VSV-infected cells express a single surface glycoprotein, VSV-G. The transport of both the wild-type and a mutant form of VSV-G, strain tsO45, has been extensively utilized to define the basic biochemical components and principles of operation of the secretory pathway (reviewed in Refs. 2, 13, and 14). In particular, tsO45 VSV-G (VSV-Gts) accumulates in the ER due to a temperature-sensitive folding defect at the restrictive temperature of 39.5 °C. Upon shift to the permissive temperature (32 °C) folding resumes, allowing us to synchronize exit from the ER. The ability to synchronize movement of VSV-Gts has played an important role in defining the normal folding intermediates associated with the molecular chaperones calnexin and BiP preceding ER export (15-18). In addition, because host protein synthesis is blocked following VSV infection of cells, VSV-Gts is the major glycoprotein transiting the secretory pathway. Therefore, VSV-Gts allows us to follow the effects of a single well defined biosynthetic cargo molecule on COPII recruitment and vesicle budding when it has accumulated in the ER as a folding intermediate or when it is subsequently matured to the folded state for transport (4, 19-23).

We have now applied a variety of techniques to monitor the effect of biosynthetic cargo on vesicle budding from secretory compartments. These include the following: (i) quantitative stereology to follow membrane flow at the ultra-structural level in vivo (24), (ii) biochemical assays to monitor ER vesicle budding in vitro (25), (iii) an assay to monitor the interactions of cargo with COPII components in the ER prior to vesicle formation (4), and (iv) an assay that allows us to follow the recruitment of COPI components to downstream pre-Golgi and Golgi compartments that may be affected by export from the ER. The effects of availability of a membrane-bound cargo molecule such as VSV-G on ER export was also compared with that of an endogenous soluble cargo molecule, the PiZ variant of alpha 1-antitrysin (PiZ) (26, 27) which accumulates in the ER under pathophysiological conditions. We now demonstrate that cargo can prevent and enhance COPII vesicle budding in vivo and in vitro. Furthermore, we show that VSV-G held in the ER interacts with the Sar1 and Sec23/24 components of the COPII machinery. We propose that the observed coupling between cargo and COPII components can modulate the formation of pre-budding intermediates that are essential for vesicle formation from the ER.

    EXPERIMENTAL PROCEDURES

Materials-- Calphostin C, Roche 31-8220, staurosporine, and phorbol 12-myristate 13-acetate were obtained from Calbiochem. Calphostin C was made fresh every 2 weeks. Both reagents were added from stock solutions in dimethyl sulfoxide (Me2SO). Digitonin was obtained from Wako BioProducts (Richmond, VA). Endoglycosidase H was obtained from Boehringer Mannheim. GS beads were obtained from Amersham Pharmacia Biotech. Other antibodies used in this study were generous gifts from the following laboratories: a polyclonal antibody against Sec23p from R. Schekman, University of California, Berkeley (Berkeley, CA); a polyclonal antibody against p58 from J. Saraste, University of Bergen (Oslo, Norway); a monoclonal antibody to VSV-G from T. Kreis (28); a monoclonal antibody against beta -COP (M3A5) from T. Kreis, University of Geneva (Geneva, Switzerland); and an anti-peptide antibody against beta -COP (EAGE) from M. Farquhar (University of California, San Diego, CA). A polyclonal antibody specific for VSV-G was described previously (22). A polyclonal antibody to Sec23 was generated against a peptide (DTEHGGSQAR) (residues 707-716) or against recombinant GST-Sec23 as described (4). The stably transfected mouse hepatoma cells (line Hep 1a) expressing recombinant PiZ variant (line H1A/RSVATZ-8) (29) or normal PiM1 human AAT (line H1A/M-15) (26) were generously provided by R. Sifers, Baylor College of Medicine (Houston).

Transmission Electron Microscopy and Morphometry-- Measurement of the basic cell morphometric parameters, the number of ER and Golgi-derived budding profiles, and the number and size of pre-Golgi intermediates were determined as described (24)

Proteins-- Recombinant Sar1 and GST-Sec23 proteins utilized in this study were prepared as described (4).

Virus Infection-- Normal rat kidney (NRK) cells were infected with VSV as described (22, 30). Briefly, cells were infected at a multiplicity of 10-20 plaque-forming units with wild-type or the tsO45 strain of vesicular stomatitis virus (21) for 4 h at the restrictive temperature. For measurement of transport in vivo, protocols were as described under "Results" and in the figure legends. For measurement of transport in vitro, preparation of cell homogenates was as described (25).

ER Budding Assay-- COPII vesicle formation reactions using NRK microsomes were performed and budding quantitated using antibodies specific for Sec23, p58, and VSV-G using Western blotting (25). To enhance preservation of the temperature-sensitive phenotype observed in vivo, in some experiments (see "Results"), 1 mM dithiothreitol was added to cells during the final 15 min of incubation at 39.5 °C. Cells were harvested, washed, and homogenized in the presence of 1 mM dithiothreitol. Subsequently, membranes were washed twice in transport buffer lacking dithiothreitol prior to freezing as described(25).

Preparation of Membranes and beta -COP Binding Reaction-- 150-mm dishes of NRK cells were washed three times with ice-cold phosphate-buffered saline and scraped with rubber policeman in a buffer containing 10 mM Hepes (pH 7.2) and 250 mM mannitol (buffer A). The cells were pelleted, resuspended in buffer A, and homogenized by passing the cell suspension 6 times through a ball bearing homogenizer (31). A post-nuclear supernatant was prepared by centrifuging the homogenate at 1000 × g for 10 min at 4 °C. 15 µl (20-40 µg protein) of post-nuclear supernatant was added to a transport reaction mixture containing 27.5 mM Hepes-KOH (pH 7.2), 2.5 mM MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM CaCl2, 1 mM ATP, 5 mM creatine phosphate, 0.2 units of rabbit muscle creatine kinase (final concentrations), and 1.5-2 mg/ml rat liver cytosol in a final volume of 200 µl on ice. The tubes were then transferred to 32 °C for 5-20 min as indicated under "Results." The reaction was terminated by transfer to ice. 1 ml of a buffer containing 25 mM Hepes (pH 7.2), 2.5 mM MgOAc, and KOAc to give the final salt concentration as indicated under "Results" was added, and the tubes were vortexed and centrifuged at 16,000 × g for 10 min at 4 °C. The supernatant was aspirated, and the tubes were centrifuged for an additional 3 min at 16,000 × g at 4 °C. The residual supernatant was removed and 25 µl of a gel sample buffer (32) added. Samples were heated for 5 min at 95 °C and resolved using SDS-polyacrylamide gel electrophoresis (32) on 7.5 or 10% polyacrylamide gels. beta -COP was detected by immunoblotting using the M3A5 monoclonal antibody specific for beta -COP and developed with alkaline phosphatase (33, 34). beta -COP was quantitated by densitometry using a Molecular Dynamics Densitometer (Sunnyvale, CA).

GST Complex Isolation-- Isolation of the pre-budding complex with GST-Sec23 was performed as described (4).

    RESULTS

The Steady-state Level of Buds on the ER Is Sensitive to Biosynthetic Cargo-- An ER-derived bud is defined as an elevation on the surface of the ER with a width of 60-80 nm that is extruded by 50% of its diameter and covered with a characteristic coat on the external leaflet of the membrane (24) (Fig. 1A, asterisks) (see "Experimental Procedures"). Such buds are found in specialized sites of the ER referred to as "transitional regions" near the Golgi (35) or "export complexes" found in the peripheral cytoplasm (24).


View larger version (106K):
[in this window]
[in a new window]
 
Fig. 1.   Release of VSV-G from retention in the ER stimulates vesicle budding in vivo. A, an electron micrograph illustrating ER budding profiles (asterisks) and vesicular-tubular (pre-Golgi) intermediates (arrowheads) involved in ER to Golgi transport (24). Bar, 0.1 µM. B, cells were either mock-infected (open bars, lanes a and c) or infected with wild-type VSV (closed bars, lanes b and d) for 4.5 h at 39.5 °C as described (30). Cells were rapidly transferred to ice and fixed (0 min (a and b)) or incubated for 5 min at 32 °C (c and d) prior to fixation. C and D, cells were either mock-infected (open bars, lanes a and c) or infected with tsO45-VSV for 4.5 h at 39.5 °C (closed bars, lanes b, d, and e). Cells were transferred to ice and fixed (0 min (a and b)), or incubated for 5 (c and d) or 30 (e) min at the permissive temperature (32 °C) prior to fixation. The number of ER buds (B and C) or pre-Golgi intermediates (D) was determined as described under "Experimental Procedures." Error bars were determined as described (24).

To assess the effect of cargo on ER export in vivo, we examined whether the number of buds found on the ER surface can be modulated by biosynthetic cargo using electron microscopy and quantitative stereology. To validate this approach, we first determined the effect of reagents that block general protein export from the ER. We have previously established using biochemical approaches that both calphostin C and okadaic acid (OKA) are potent inhibitors of the export of the type 1 transmembrane protein vesicular stomatitis virus glycoprotein (VSV-G) from the ER, whereas phorbol 12-myristate 13-acetate is stimulatory (36, 37). The effects of these reagents on the formation of ER buds as assessed by quantitative morphometry paralleled those seen in previous biochemical assays. In control cells, we detected on average 248 ± 25 buds per cell at steady state. Treatment of cells with OKA reduced by >95% the number of budding structures on the ER (12 ± 5 buds per cells). This result is similar to the recently reported morphological effects of OKA on export of the pre-Golgi intermediate marker protein p53/58 from the ER (38). Calphostin C led to an 85% reduction (40 ± 10 buds per cell, respectively), whereas phorbol 12-myristate 13-acetate treatment resulted in a 2-fold increase in their number (480 ± 11). Changes in ER export also led to corresponding alterations in the abundance and size of downstream pre-Golgi intermediates. These structures are composed morphologically of clusters of vesicles and tubules (Fig. 1A, arrowheads) (20, 24, 39, 40) (see "Experimental Procedures"). In the absence of inhibitors, cells contained on average 67 ± 7 pre-Golgi intermediates at steady state. This value was reduced to 4 ± 1 and 14 ± 4 in the presence of OKA and calphostin C, respectively.

By having established that the number of ER buds scored by quantitative morphometry serves as a reliable measure of protein and membrane flow from the ER through the secretory pathway, we next followed the effect of a single biosynthetic cargo species on ER export. For this purpose, we infected cells with the tsO45 strain of vesicular stomatitis virus (VSVts) which encodes a temperature-sensitive mutant of the type 1 transmembrane surface glycoprotein VSV-G (VSV-Gts). When expressed at the restrictive temperature (39.5 °C), VSV-Gts accumulates in the ER due to a folding defect (41). Accumulation in the ER at 39.5 °C is not a consequence of aggregation as VSV-Gts is freely diffusible (42). Because virus infection inhibits host protein synthesis (43), this approach allows us to simultaneously reduce endogenous biosynthetic cargo and introduce a single major cargo species into the ER. Upon shift to the permissive temperature (32 °C), VSV-Gts exits the ER via COPII vesicular carriers (19, 21, 22, 25). If the availability of cargo for export can influence the formation of COPII-derived vesicular carriers in vivo, then we should detect differences between the number of ER buds and downstream intermediates found at the restrictive and permissive temperatures.

We first examined the export of wild-type VSV-G to rule out the trivial concern that viral pathogenesis may interfere in some unexpected way with the appearance of budding structures. Budding activity (as defined by the number of budding profiles per µm2 ER membrane surface) in mock-infected or wild-type-infected cells at 39.5 °C was generally 2-2.5-fold greater than that observed at 32 °C (Fig. 1B). The abundance of pre-Golgi intermediates in mock-infected NRK cells was also greater (~1.8-fold) at 39.5 °C than at 32 °C (Fig. 1D, compare lane a to lane c), corresponding to the enhanced level of ER export. Consistent with this result, the rate of processing of wild-type VSV-G oligosaccharides by Golgi-associated enzymes was ~2.5-fold faster at 39.5 °C than at 32 °C (data not shown). Thus, the transport activities observed in control and wild-type virus-infected cells were comparable, and in both cases, budding activity was temperature-dependent.

In contrast to the lack of effects of wild-type VSV-G on budding when compared with mock-infected cells, the number of ER buds in tsO45-infected cells retained at 39.5 °C was nearly 3-4-fold lower (a 65-75% decrease) (Fig. 1C, lane b) than that observed in mock-infected (Fig. 1C, lane a) or wild-type VSV-infected cells (Fig. 1B, lane b). No changes in any of the basic cell morphometric parameters (volume and surface area) were detected under these conditions (not shown). Although the surface area and volume of the ER in tsO45-infected cells held at 39.5 °C increased only slightly (~5%), we observed an ~50% reduction in the number of pre-Golgi intermediates (Fig. 1D, lane b), a 40% (± 5%) decrease in the size of the Golgi stack, and a 60% (± 10%) reduction in the number of Golgi buds relative to mock-infected cells. These results suggest that the reduction in ER budding activity observed in tsO45-infected cells held at 39.5 °C (Fig. 1C, lane b) results in a corresponding reduction in size and budding activity of downstream organelles.

Because export from the ER appears to be sensitive to cargo, we would predict that transfer of tsO45-infected cells to 32 °C should restore budding activity and membrane flow. Indeed, a 5-min shift to 32 °C led to a wave of export activity, with a 1.5-fold increase (Fig. 1C, lane d) in vesicle budding over that observed in mock-infected cells held at 32 °C (Fig. 1B, lane c). This wave of export was reflected in a corresponding ~1.4-fold increase in pre-Golgi intermediates (Fig. 1D, lane d) and an ~1.6-fold increase in Golgi size and the number of Golgi-associated buds (not shown). This value returned to the steady-state level seen in control cells within 30 min (Fig. 1C, lane e). We conclude that a major biosynthetic cargo molecule such as VSV-Gts can affect general membrane flow from the ER by preventing or enhancing ER budding.

The Availability of Soluble Biosynthetic Cargo Affects COPII Vesicle Formation-- The effects of VSV-Gts, a transmembrane protein, on export led us to also characterize the potential role of accumulation of an endogenous soluble protein, in this case the human PiZ variant of alpha 1-antitrysin (AATPiZ) (27). Unlike wild-type monomeric alpha 1-antitrypsin (AATwt), which is secreted rapidly from the ER, greater than 85% of the transport-impaired PiZ variant is retained within the ER and is subjected to rapid intracellular degradation (29). In a subset of PiZ variant carrier patients, the degradation of the protein is markedly reduced leading to its accumulation in the ER (27). These patients develop severe liver injury, a problem that may potentially reflect a more global deficiency in ER and/or cell function. Interestingly, when we examined isogenic cell lines expressing either the wild-type alpha 1-antitrypsin (ATTwt) or those accumulating the PiZ variant (ATTPiZ), we found an ~30% decrease in the number of ER buds in the PiZ variant (Fig. 2A, compare lane a to lane e). Thus, steady-state accumulation of ATTPiZ in the ER led to reduced levels of budding profiles.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   ER buds are significantly reduced in response to accumulated PiZ variant in vivo. A, the abundance of ER budding profiles in a stably transfected hepatoma cell line (Hepa 1A) expressing the wild-type (black bars) or PiZ variant (gray bars) in the absence (0 h) or after increasing time of treatment with CHX. Quantitation was performed as described under "Experimental Procedures." B (open squares), NRK cells infected with tsO45 VSV at 39.5 °C for 4 h were radiolabeled at 39.5 °C and chased at 32 °C in the presence of unlabeled methionine as described (30). Closed circles, radiolabeled cells as described above were incubated for an additional 4 h in the presence of CHX (50 µg/ml) prior to transfer to 32 °C. The fraction of VSV-G processed to Golgi-modified forms was determined as described (30). C, the abundance of ER-budding profiles (black bars) were measured in vivo in non-infected NRK cells in the absence (0 h) or after increasing time (up to 4 h) in the presence of CHX (50 µg/ml). Bars indicate standard error of the mean for three independent experiments.

To mimic the more exaggerated long term accumulation of alpha 1-antitrypsin found in PiZ variant patients, we took advantage of previous studies which have demonstrated that ER degradation of the PiZ variant requires protein synthesis (44). Addition of CHX not only prevents degradation of the PiZ variant but also reduces the content of other cargo proteins in the ER. As such, this condition leads to a temporary relative increase in the abundance of polymerized AATPiZ variant in the ER (44, 45). In addition, incubation of cells in the presence of CHX allows us to examine independently the role of general cargo reduction on ER-budding structures. This is an important control in the case of VSV-infected cells where the virus inhibits host protein synthesis. Previous studies in yeast have shown that CHX treatment does lead to a partial reduction of ER-derived vesicles (46).

To test first for any potential nonspecific effects of CHX on transport, we examined whether exposure of tsO45 virus-infected cells to CHX for up to 4 h at the restrictive temperature alters the subsequent kinetics of ER to Golgi transport of VSV-Gts at the permissive temperature in the presence of the drug. As shown in Fig. 2B, VSV-G accumulated at the restrictive temperature for 4.5 h was radiolabeled at 39.5 °C and incubated in the presence of CHX for an additional 4 h prior to transfer to 32 °C. Strikingly, no inhibitory effect was observed compared with the transport kinetics of the control that was not incubated for an additional 4 h in the presence of CHX. A similar lack of effect was observed when wild-type virus-infected cells were treated up to 2.5 h with CHX, and the rate of transport was assessed immediately following removal of the drug (data not shown). These results led us to conclude that ER to Golgi transport factors are stable following exposure to CHX for up to 4 h.

Because rapidly transported cargo such as the soluble molecule albumin or VSV-G require ~3 h to be effectively drained from the ER in the presence of CHX (37, 47, 48) (data not shown), whereas many other cargo molecules exit the ER with considerably longer half-times (49, 50), we anticipate that treatment with CHX for up to 4 h should only have partial effects on the formation ER-derived buds. Consistent with this prediction, only an ~30% reduction was observed after a 4-h treatment of uninfected NRK cells (210 ± 20 buds per cell (minus CHX); 130 ± 15 buds per cell (plus CHX)) (Fig. 2C). The number of buds in wild-type VSV-infected NRK cells was reduced similarly (375 ± 20 buds per cell (minus CHX) to 190 ± 15 buds per cell (plus CHX)), emphasizing that viral-infected cells behave identically to uninfected cells. Identical results were observed in rat basophilic leukemia cells treated with CHX (data not shown). When AATwt-expressing cells were treated with CHX, we also observed a modest ~25-30% reduction in budding profiles (Fig. 2A, black bars, compare lane a to lane d). In all of these controls, the reduction in the number of detectable ER buds was not a consequence of slower transport leading to flattening of the budding profile, as the mean height distribution of residual buds did not change (62 ± 22 nm (minus CHX)), 63 ± 30 nm (plus CHX)), and inhibition was fully reversible.

In contrast to the above results, the effects of CHX on AATPiZ-expressing cells were more dramatic, leading to an ~45-50% decrease in budding profiles (Fig. 2A, gray bars, compare lane e to lane f) (44, 51). When compared with the AATwt-expressing cells, this level of accumulation resulted in a 60% decrease in budding (Fig. 2A, compare lane a to lane h). Thus, while the partial (~30%) reduction of budding in response to general cargo reduction by treatment of cells with CHX is consistent with its effects on vesicle formation in vivo in yeast (46), it cannot account for the nearly 2.5-or 4-fold reduction observed during following accumulation of the PiZ variant in the presence of CHX (Fig. 2A) or VSV-Gts at the restrictive temperature (Fig. 1), respectively.

Release of COPII-coated Vesicles in Vitro Is Linked to VSV-G Export-- Given the compelling effects of VSV-Gts on ER export in vivo, we examined whether these events can be reconstituted in vitro to study its potential mechanism biochemically. For this purpose, we utilized a budding assay that reconstitutes the release of VSV-Gts into COPII-coated vesicles from mammalian microsomes (25). In brief, to follow vesicle budding in vitro a post-nuclear supernatant fraction is prepared from homogenates of cells infected at 39.5 °C with tsO45 virus to accumulate VSV-Gts exclusively in the ER. Following washing to remove cytosolic components, membranes are incubated in a transport mixture containing cytosol and an energy source in the form of ATP. The fraction of VSV-Gts exported from the ER is measured using differential centrifugation to separate more rapidly sedimenting ER and Golgi membranes that are recovered in a medium speed pellet (MSP) from slowly sedimenting ER-derived COPII vesicles that remain in the medium speed supernatant (MSS). The amount of VSV-Gts in each fraction can be quantitated using SDS-polyacrylamide gel electrophoresis and Western blotting (25).

Fig. 3 illustrates that the in vitro budding reaction fully reconstitutes the temperature-sensitive export of VSV-Gts observed in vivo. At early time points at 32 °C, a transient peak of VSV-Gts (~20% of total VSV-G) appears in COPII vesicles in the MSS (Fig. 3, open squares). At later time points this pool declines and is recovered in the MSP. We have previously demonstrated that the appearance of VSV-G in the MSP is concomitant with processing of VSV-Gts to Golgi-modified forms (25). In contrast, incubation of microsomes at 39.5 °C strongly (>80%) inhibited export of VSV-Gts from the ER (Fig. 3, closed squares), consistent with the fact that under these conditions VSV-Gts was not processed to Golgi-modified forms (data not shown).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 3.   Release of VSV-G containing vesicles in vitro is blocked at the restrictive temperature. Microsomes prepared from tsO45 VSV-infected cells were incubated at 32 °C (open squares) or 39.5 °C (closed squares) for the indicated times, and the release of VSV-G-containing COPII vesicles from the ER found in the medium speed pellet (MSP) (P in upper panel) to the medium speed supernatant (S in upper panel) was determined by Western blotting as described (25). The Western blot (upper panel) was quantitated (lower panel) as described under "Experimental Procedures."

Because the release of VSV-Gts from ER microsomes was inhibited at the restrictive temperature, we examined whether other proteins that exit the ER were similarly affected. We have previously shown that p58, a transmembrane protein that actively recycles between the ER and pre/cis-Golgi elements (19, 25, 40), is a component of COPII vesicles (25). p58 does not bind VSV-G at either the permissive or restrictive temperatures (data not shown), is not required for VSV-G export from the ER (52), and therefore serves as an independent marker for COPII vesicle formation.

We examined the rate and extent of release of p58 into the MSS at the permissive and restrictive temperatures in mock- and tsO45 VSV-infected cells. In mock-infected cells, incubation at 39.5 °C led to a stimulation of p58 recovery in COPII vesicles over that observed at 32 °C (Fig. 4A, dotted lines; closed squares (39.5°), open squares (32 °C)). Similar results were observed using microsomes prepared from wild-type-infected cells (not shown). In contrast, export of p58 in microsomes prepared from tsO45 VSV-infected cells was markedly reduced at 39.5 °C compared with 32 °C (Fig. 4A, solid lines; closed circles (39.5°), open circles (32 °C)). Up to 70-80% inhibition was observed at the peak of p58 release. These in vitro results are consistent with our stereological analysis of budding in vivo (Fig. 1) and provide independent biochemical evidence that the export of at least one endogenous component of the transport machinery is responsive to cargo regulation of COPII vesicle formation.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   General ER budding in vitro is blocked at the restrictive temperature. A, microsomes prepared from mock-infected (MI) or tsO45 VSV-infected cells (I) were incubated at the permissive (32 °C) or restrictive (39.5 °C) temperatures for the indicated times, and the release of p58 from the ER found in the medium speed pellet (MSP) (P in upper panels) to the medium speed supernatant (MSS) (S in upper panels) was determined by Western blotting (upper panels) and quantitated (lower panel) as described under "Experimental Procedures" (25). The export of p58 in mock-infected cells is indicated by broken lines; export in virus-infected cells is indicated by solid lines. B, microsomes prepared from tsO45-VSV-infected cells were incubated at 32 or 39.5 °C for 10 min, and the appearance of vesicles coated with Sec23 in the MSS was determined by Western blotting using an antibody specific for Sec23 as described under "Experimental Procedures."

If the formation of ER-derived COPII vesicles is linked to cargo availability as suggested by the above experiments, one would anticipate that the general release of COPII vesicles from tsO45 VSV-infected cells would be inhibited at 39.5 °C. Therefore, we analyzed the effect of cargo availability on COPII budding from the ER by following the recovery of the membrane-bound COPII component Sec23 in the MSS of our budding assay. In controls, when microsomes prepared from mock-infected cells were incubated at 39.5 °C, we observed a significant stimulation (~1.5-fold) of the release of Sec23-containing COPII-coated carriers over that observed at 32 °C (Fig. 4B, compare lane a to lane b). A similar result was obtained using wild-type virus-infected cells (not shown), values reflecting the temperature sensitivity of ER budding in vitro (25). In contrast, microsomes prepared from tsO45-infected cells, when incubated at 39.5 °C, exhibited an ~2.5-fold decrease in Sec23 abundance in the MSS compared with 32 °C (Fig. 4B, compare lane c and lane d), and an ~4-fold decrease compared with microsomes prepared from mock-infected cells (Fig. 4B, compare lane d to lane b). These results are consistent with our stereological analysis of ER budding in vivo (Fig. 1) suggesting that the level of transportable biosynthetic cargo is coupled to the general formation of COPII vesicular carriers.

The Activated Form of Sar1 Promotes Stable Coat Assembly but Will Not Promote COPII Vesicle Formation in the Absence of Available Biosynthetic Cargo-- Under typical incubation conditions, the assembly and disassembly of COPII coats on membranes is regulated by the GTPase cycle of the Sar1 GTPase (19, 25, 53). In our microsome-based assay, the release of Sec23-containing COPII vesicles into the MSS occurs with a transient peak at ~10 min (Fig. 5A, open circles). In this case, the abundance of Sec23 in the MSS rapidly declines to background levels, reflecting the fact that COPII coats are rapidly lost from vesicles (19, 25, 53). To determine whether the formation of Sec23-containing vesicles was indeed dependent on COPII coat assembly, we supplemented the assay with the GDP-restricted mutant of Sar1 (Sar1[T31N]) (to be referred to as Sar1-GDP) which inhibits the activation of endogenous Sar1 in the reaction and prevents the release of both VSV-Gts and p58 into the MSS (25). As shown in Fig. 5A (open squares), it also blocks the general release of Sec23-coated carriers. Thus, Sar1-dependent budding recapitulates budding events observed in vivo.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 5.   Activated Sar1-GTP does not promote exit of VSV-Gts from the ER. Microsomes prepared from mock-infected cells were incubated for the indicated times with no additions (open circles), in the presence of the Sar1-GDP-restricted mutant (Sar1[T39N]) (open squares), or the Sar1-GTP-restricted mutant (Sar1[H79G]) (closed squares), and the abundance of COPII vesicles released into the MSS was determined by Western blotting for the COPII coat subunit Sec23 as described under "Experimental Procedures." Recombinant mutant proteins (1 µg) were prepared as described (85). B, microsomes prepared from infected cells were incubated for 10 min at 32 (a and b) or 39.5 °C (c and d) in the absence (a and c) or presence (b and d) of 1 µg Sar1-GTP, and the amount of Sec23-bound vesicles was recovered in the MSS determined by Western blotting. C, microsomes prepared from infected cells were incubated for 10 min at 32 (a and d) or 39.5 °C (b, c, e and f) in the absence (a, b, d, and e) or presence (c and f) of 1 µg of Sar1-GTP, and amount of VSV-G or p58 released into the MSS was determined by Western blotting. Typical results of three independent experiments are shown.

Given the importance of Sar1 activation in cargo export, we examined whether a pre-activated form of Sar1 could bypass the block in ER-derived vesicle formation imposed by incubation of ER prepared from VSVts-infected cells at 39.5 °C. For this purpose, we supplemented the assay with Sar1[H79G] (19), a mutant that is restricted to the GTP-bound state (to be referred to as Sar1-GTP). We have previously shown that the Sar1-GTP-restricted mutant does not further stimulate the rate or extent of formation of vesicles containing VSV-Gts or p58 at 32 °C (25). However, we did find that in the presence of the Sar1-GTP mutant, VSV-G/p58 released into COPII vesicles at 32 °C accumulates in the MSS as the delivery of vesicles to the Golgi is largely blocked in response to the inability of coats to be disassembled (19, 25).

When we examined the effect of Sar1-GTP on general vesicle recovery in the MSS at 32 °C using Sec23 as a marker, we observed an ~3-fold increase over incubations performed in the absence of the activated mutant (Fig. 5A, compare closed squares to open circles), reflecting the stabilization of the COPII coat on vesicle membranes by the activated GTPase. The addition of Sar1-GTP at 39.5 °C led to a similar 3-fold increment in the recovery of Sec23 in the MSS (Fig. 5B, compare lane c to lane d) when compared with the effects of Sar1-GTP at 32 °C (Fig. 5B, compare lane a to lane b). Again, this reflects the stabilization of the COPII coat to vesicles formed at the higher temperature. Strikingly, the addition of Sar1-GTP to the assay did not lead to either VSV-Gts (Fig. 5C, compare lane b to lane c) or p58 release into the MSS at 39.5 °C (Fig. 5C, compare lane e to lane f) and therefore has no effect on the extent of general vesicle release. Thus, addition of an activated form of Sar1 was not sufficient to by-pass the absence of a transportable form of biosynthetic cargo in the ER, emphasizing the importance of VSV-Gts in regulating these events.

VSV-G Can Interact with the COPII Machinery in the Unfolded State-- Accumulation of VSV-Gts in the ER appears to play a dominant inhibitory role in the formation of COPII vesicles. Because in viral-infected cells VSV-Gts is the major form of cargo for export, it may have a previously unsuspected interaction with the COPII budding machinery even at the restrictive temperature. To examine this possibility, we took advantage of recent studies where we demonstrated that under conditions that do not support vesicle budding from the ER, VSV-Gts is selected for ER export by interacting with Sec23 or the Sec23/24 complex in a Sar1-dependent manner (4). In these studies, we demonstrated that incubation of VSV-Gts containing ER membranes at the permissive temperature in the presence of glutathione S-transferase-tagged Sec23 (GST-Sec23) can lead to the efficient recovery of VSV-Gts in a detergent-soluble pre-budding protein complex on glutathione-Sepharose beads (GS beads). Recovery on GS beads is Sar1A-dependent and does not occur when membranes are retained on ice (4). Moreover, under these conditions, the VSV-Gts/Sec23-containing complex excludes ER resident proteins such as BIP, calnexin, and ribophorin II, indicative of efficient sorting from resident ER proteins (4). On the average, 10-20% of the total soluble VSV-G can be recovered on GS beads at 32 °C (4).

To determine whether a pre-budding complex containing VSV-Gts and COPII components can be detected at the restrictive temperature, membranes were washed to remove any residual bound Sar1, Sec23/24, and Sec13/31 complex (4). Washed membranes remain export-competent and retained their temperature sensitivity as incubation with cytosol and Sar1-GTP led to the accumulation of VSV-G-containing vesicles at 32 °C but not at 39.5 °C (Fig. 6A). To determine when cargo becomes associated with the Sec23 partial coat, microsomes incubated at 32 or 39.5 °C with recombinant GST-Sec23 and/or Sar1A mutants were pelleted, washed, and solubilized in a detergent containing buffer. Following centrifugation to remove insoluble material, the supernatant was incubated with glutathione-Sepharose (GS) beads, and bound GST-Sec23 was quantitated using Western blotting (4). As shown in Fig. 6B, recovery of VSV-Gts or Sec23 was not observed in samples held on ice (Fig. 6B, lane a). Strikingly, the level of recovery of VSV-Gts on GS beads at the restrictive temperature (39.5 °C) was similar to the level of recovery at the permissive temperature (32 °C) (Fig. 6B, upper panel, compare lane b to lane d). Recovery was specific for the COPII as it was blocked by incubation with the inactivated, Sar1-GDP mutant (Fig. 6B, lanes c and e). Moreover, in both cases, the recruitment of VSV-Gts was directly correlated with the recruitment of GST-Sec23 (Fig. 6B, lower panel, compare lanes b to c and lanes d to e). Thus, although budding may be largely blocked at 39.5 °C in vitro (Fig. 3), recruitment of the COPII component Sec23 and VSV-Gts to a pre-budding complex is not. These results support the interpretation that misfolded VSV-G can sequester the COPII budding machinery. By establishing conditions to control the export of a major biosynthetic cargo species from the ER, we have identified a novel intermediate preceding completion of protein folding.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   VSV-G can be detected in a complex with Sec23 at 39.5 °C. A, salt-washed microsomes prepared as described under "Experimental Procedures" were incubated at 32 °C (a) or 39.5 °C (b) for 30 min in the presence of rat liver cytosol and 1 µg of the activated Sar1A (GTP-restricted) mutant. The amount of VSV-Gts (percent of total) released from the ER in COPII vesicles was determined as described (25). B, salt-washed microsomes were incubated on ice (a), at 32 (b and c), or 39.5 °C (d and e) for 30 min with GST-Sec23 in the absence (a) or presence of either the GTP-restricted (b and d) or GDP-restricted (c and e) Sar1 mutant. Membranes were pelleted, solubilized, and incubated with GS beads, and the amount of GST-Sec23 or VSV-Gts recovered on beads was determined using Western blotting as described under "Experimental Procedures." Typical results of three independent experiments are shown.

Biosynthetic Cargo Reduction Leads to Changes in Vesicle Budding from Downstream Compartments-- The substantive effects of VSV-Gts on ER budding in vivo and in vitro (Fig. 1 and Fig. 4) and its ability to sequester COPII components at the restrictive temperature (Fig. 6) led us to assess whether biosynthetic cargo availability could be correlated with the function of downstream secretory compartments directing recycling of transport components. Consistent with this possibility, we had already noted a decrease in vivo in the abundance of pre-Golgi intermediates in tsO45-infected cells held at the restrictive temperature (Fig. 1). In addition, using indirect immunofluorescence, we have found that the pre-Golgi intermediate marker protein p58 as well as the COPI subunit beta -COP, a coat complex involved in recycling from both pre-Golgi and Golgi compartments (2, 54-56), undergo a significant change in their morphological distributions in response to infection at the restrictive temperature or treatment with CHX. Whereas p58 migrates from its typical pre-Golgi and Golgi punctate distribution (19, 22, 39, 40) to the ER, COPI shifts from its typical peri-nuclear membrane-bound Golgi localization (19, 54) to a diffuse cytosolic localization (data not shown). These qualitative observations raised the possibility that reduced membrane flow from the ER results in a decreased need for COPI function in recycling. We therefore examined the ability of microsomes containing pre-Golgi and Golgi membranes to recruit COPI quantitatively as a more direct measure for these events.

Previous studies have shown that incubation of Golgi membranes in the presence of cytosol, ATP, and GTPgamma S, a poorly hydrolyzable analog of GTP which prolongs the activation of the small GTPase ARF1, enhances and stabilizes the recruitment of COPI to pre- and cis-Golgi elements (19, 57-60). Unlike COPI present on membranes prior to activation of ARF1, the COPI pool actively engaged in transport is resistant to release by treatment with high salt (61, 62). We utilized this difference to quantitate the steady-state recruitment of activated COPI in vitro to membranes.

We first examined the recruitment of COPI to control (uninfected) microsomes. We found that COPI present on microsomes held on ice can be removed by a high salt wash (250 mM KOAc) indicative of a low affinity, inactive form (Fig. 7A, open circles). In contrast, when microsomes were incubated in the presence of cytosol, ATP, and GTPgamma S for 5 min at 32 °C to maximize the ARF1 activation (19), beta -COP was now efficiently retained by membranes following the high salt wash (Fig. 7A, closed circles). Further incubation did not lead to an increase in COPI binding. As an additional control, we examined the ability of CHX-treated cells to recruit the high salt-resistant form of COPI in the absence GTPgamma S to assess recruitment in response to a partial cargo reduction. At low salt concentrations (75 mM KOAc) CHX-treated and control cells retained identical levels of beta -COP (Fig. 7B). Incubation of control membranes for 5 min in vitro in the absence of GTPgamma S showed recruitment of a pool of COPI of which 40-60% of the total membrane-associated pool of COPI was in the high salt-resistant COPI form (Fig. 7B, open circles). In contrast, microsomes prepared from CHX-treated cells had a 3-4-fold reduced level of membrane-associated COPI following the high salt wash in the absence of GTPgamma S (Fig. 7B, closed circles). The observed deficiency in the recruitment of the high salt-resistant form of COPI was not a consequence of the inability to activate the ARF1 pathway in CHX-treated cells. When these microsomes were incubated for 5 min in the presence of GTPgamma S, COPI coats resistant to high salt extraction were recruited to statistically comparable levels to those observed in control membranes (Fig. 7B, lanes a and b). These results suggest that COPI binding is a sensitive measure of Golgi function in response to reduced cargo in the pathway.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7.   Depletion of cargo interferes with the recruitment of beta -COP to membranes. A, microsomes prepared from cell homogenates of NRK cells were maintained on ice (open circles) or were incubated for 5 min at 32 °C in the presence of an ATP-regenerating system, cytosol, and GTPgamma S (100 µM) (closed circles) as described under "Experimental Procedures." Membranes were washed with the indicated concentration of KOAc, and the fraction of beta -COP remaining associated with membranes was determined by Western blotting as described under "Experimental Procedures." Values are reported as percent of beta -COP binding relative to the level of beta -COP attached to membranes following the 75 mM KOAc salt wash in cells treated with GTPgamma S (maximal binding). B, microsomes prepared from control (open circles) or CHX-treated (4 h) (closed circles) cells were incubated for 5 min in the presence of an ATP-regenerating system and cytosol in the absence of GTPgamma S. Values are reported as percent of beta -COP binding relative to the level of beta -COP present on membranes following the 75 mM KOAc salt wash. Bars a and b illustrate the effect of the presence of 100 µM GTPgamma S on beta -COP recruitment in control (a) and CHX-treated (b) membranes. C, microsomes prepared from cell homogenates of mock-infected (squares) or tsO45 VSV-infected (closed circles) were incubated in vitro for 5 min at the restrictive temperature (39.5 °C) as described under "Experimental Procedures." Membranes were washed with the indicated concentration of KOAc and the fraction (percent) of beta -COP remaining associated determined as described above. D, microsomes prepared from mock-infected (open squares) or tsO45 VSV-infected (circles) cells were incubated for 5 (closed circles) or 20 min (open circles) in the presence of ATP-regenerating system and cytosol. A-D, the data presented are the average of two or more experiments with the error bar indicating the standard error of the mean.

We next examined the response of downstream compartments to regulated release of VSV-Gts from the ER. We first tested whether a reduction in transported cargo in microsomes prepared from cells infected with the tsO45 virus at 39.5 °C would lead to a decrease in COPI binding. Microsomes prepared from mock-infected cells incubated for 5 min at 39.5 °C efficiently recruited the high salt-resistant form of COPI (Fig. 7C, open squares). In contrast, recruitment of the high salt-resistant form of COPI was 3-4-fold lower in microsomes prepared from tsO45-infected cells incubated for 5 min at 39.5 °C (Fig. 7C, closed circles) and did not increase upon further incubation (not shown). When these microsomes were incubated at 32 °C for 5 min, no recruitment was observed (Fig. 7D, closed circles), whereas after incubation for 15-20 min, high salt-resistant COPI binding was comparable to that observed in microsomes prepared from mock-infected control cells (Fig. 7D, open circles and squares). The lag period observed in the recruitment of high salt-resistant forms of COPI at 32 °C by microsomes prepared from infected cells is consistent with the time period (~15-20 min) we have previously shown to be required to efficiently mobilize VSV-Gts to pre-Golgi intermediates in semi-intact cells (19, 20, 22). Thus, the observed changes in the recruitment of COPI is consistent with the ability of cargo to modulate COPII vesicle formation through its recruitment to pre-budding intermediates.

    DISCUSSION

Biosynthetic cargo exiting the ER includes a wide collection of newly synthesized molecules, protein and lipids, that are mobilized from the ER to distinct cellular and extracellular destinations. Other forms of cargo incorporated into COPII vesicles also includes proteins that are continuously recycled between ER and Golgi compartments. Recycling proteins include transport machinery proteins involved in cargo selection, in vesicle formation, and in targeting and fusion (reviewed in Refs. 55 and 56). These proteins, like biosynthetic cargo, are packaged into the budding vesicle by interacting either directly or indirectly with COPII components (4, 5, 63). Given this complexity, we introduced one cargo molecule, VSV-G, into the ER. Due to the ability of VSV to inhibit host-protein synthesis, VSV-G becomes the major cargo mobilized through the secretory pathway. Moreover, the transport of VSV-G has been extensively characterized and used to define the basic components and principles of the secretory pathway (2, 13). In non-infected cells, biosynthetic cargo accumulation and its potential effects on vesicle budding will be tightly controlled by activation of the ER degradation machinery (64) and, possibly, the ER overload response (65). In contrast, in VSV-infected cells these checkpoints are largely bypassed through the ability of the virus to prevent host protein synthesis, allowing us to accumulate VSV-Gts in the ER in a fully functional form. This experimental system, as with many biochemical approaches, represents an exaggeration of the normal course of cargo-related events in the ER. However, it provides us with a unique window to examine the relationship between a defined and controllable form of cargo and the existence of novel intermediates involved in COPII vesicle budding.

We have presented in vivo evidence using quantitative morphometry that the availability of biosynthetic cargo in the ER can both prevent and enhance the formation of COPII vesicles that mediate ER export. This effect was reconstituted in vitro by following the movement of two separate markers for ER export: the COPII component Sec23, a marker for general vesicle formation, and the endogenous recycling protein p58, which is transported independently of VSV-G. Although speculated to be a general mannose-binding lectin, (66), it does not form a complex with VSV-G.2 Moreover, recent studies now suggest that p58, a major component of COPII vesicles (25, 52), may have a selective role in transport (67, 68). A strong correlation was observed between the export of p58, the formation of COPII vesicles in vitro, and COPII vesicle formation in vivo as measured using quantitative stereology in response to VSV-Gts. These observations support our conclusion that a single biosynthetic cargo molecule such as VSV-G can modulate general COPII vesicle budding. Importantly, the inhibition of budding observed during accumulation of cargo in the ER was readily reversible. The folding of VSV-Gts following transfer to the permissive temperature led to rapid and efficient stimulation of COPII vesicle production. This important result indicates that the increased availability of VSV-G led to efficient coupling to the COPII machinery. Moreover, these results negate the possibility that the accumulation of cargo in a non-transportable form had an indirect effect by irreversibly "poisoning" the budding machinery in vivo or in vitro. Because biosynthetic cargo can have a dominant effect over the movement of an endogenous component such as p58, it is apparent that general vesicular traffic is tightly modulated in virus-infected cells. Whether VSV-G can exert a similar dominant effect on other biosynthetic cargo molecules remains to be tested.

Given that virus infection inhibits host protein synthesis, as controls we examined the effects of CHX on the level of ER budding profiles in vivo. In these experiments, although we found that treatment with CHX for up to 4 h had no general effect on the kinetics of ER to Golgi transport, treatment did lead to a reduction in ER budding profiles of ~30%. It is apparent that the decrease observed by cargo reduction alone cannot account for the much larger 2.5-4-fold effects of the accumulation of cargo in the ER observed with VSV-Gts or the PiZ variant of alpha 1-antitrypsin. However, we cannot exclude the possibility that the reduced budding observed in the presence of CHX (and therefore a reduced load of readily available cargo) supports our observations with VSV-G that normal biosynthetic cargo plays role in ER export.

The limited decrease in budding in response to the treatment with CHX only may only reflect the loss of export activity of rapidly transported endogenous cargo such as albumin. Many other proteins, including cell-surface receptors and soluble proteins, are exported at significantly slower rates from the ER (49, 50). Alternatively, in vivo, a pool of recycling components involved in maintaining the function of downstream pre-Golgi and Golgi compartments may be in continuous flux through the ER. Short term treatments with CHX, such as used in the present experiments, would be expected to have only a minor effect on the transport of these proteins. Our results with CHX showing a partial reduction in COPII budding in mammalian cells are agreement with those obtained in in vivo studies in yeast in response to CHX (46). However, in vitro studies in yeast using an assay that reconstitutes ER budding could not reproduce this effect, leading to the suggestion that cargo recruitment is uncoupled from vesicle formation (69). This in vitro result is now consistent with the more recent observations by Schekman and colleagues (70) that purified yeast COPII components can self-assemble on liposomes and yeast ER membranes on ice to generate empty coated vesicles. This degree of uncoupling of assembly of the COPII coat from other endogenous components therefore may markedly limit the sensitivity of in vitro assays to detect the effects of modest cargo reduction observed in CHX-treated membranes.

Recently, in vitro binding experiments using purified COPII components has led to the suggestion that coat assembly is promoted by snap- receptor (SNARE) components and that this may be key in vesicle formation (71). In contrast, we find that COPII recruitment and vesicle formation under physiological conditions is largely sensitive to the availability of biosynthetic cargo. Moreover, genetic studies to date suggest that SNARE components largely function in vivo following vesicle formation (72, 73). Whereas an explanation for these apparently contradictory findings between in vivo and in vitro studies on SNARE function remains to be resolved, if we view SNARE components as essential cargo whose recruitment needs to be physiologically coordinated with biosynthetic cargo, then these results and our in vivo and in vitro results are strongly consistent with a potential general role for many forms of cargo in modulating COPII vesicle formation.

To provide additional evidence that membrane flow from the ER can be controlled by biosynthetic cargo, we found that both the reduction and stimulation of ER export was proportionally conserved in all of the immediate downstream intermediates. These include changes in the general abundance of pre-Golgi intermediates, budding profiles on the CGN, and the size and volume of the Golgi stack in vivo in virus-infected cells held at the restrictive temperature to retain VSV-Gts in the ER. We have observed similar modest effects following a 4-h treatment with CHX,3 results in agreement with studies by Howell and colleagues (74). Moreover, we also observed a corresponding decrease in the ability of residual pre-Golgi and Golgi compartments to recruit COPI coats in vivo and in vitro. This may reflect the expected loss in the need for retrograde recycling of ER-derived components or, potentially, for bi-directional transport as COPI has been suggested to promote anterograde transport through the Golgi stack (75). In support of the possibility that ER export affects the function of downstream compartments, a recent study has demonstrated that a recycling soluble cargo molecule that contains the retrograde sorting determinant KDEL is required for activation of the Golgi-localized receptor ERD2 to initiate retrieval to the ER (76, 77). In this case, activation has been proposed to modulate ARF1 GTPase activity, which, in turn, controls COPI recruitment and the function of ERD2 in retrieval. Collectively, the combined results demonstrate a functional relationship between the availability of ER biosynthetic cargo and the level of membrane traffic between subsequent downstream secretory compartments.

To account for the observed changes in COPII vesicle formation in response to the availability of biosynthetic cargo, we analyzed the ability of VSV-G that was accumulated in the ER to interact with the COPII components found in pre-budding complexes (4). To our surprise, we observed comparable VSV-G recovery in Sar1/Sec23 containing pre-budding complexes at both the transport permissive (32 °C) and transport-impaired (39.5°) temperatures. This unexpected association of misfolded cargo with the ER export machinery is compatible with the previously reported low, yet detectable, transport of misfolded cargo proteins such as VSV-Gts at the restrictive temperature. Such transport is observed in vivo only when excessive infection conditions are used to saturate the folding pathway, leading to export of monomeric VSV-G and its subsequent retrieval from pre-Golgi intermediates through its interaction with the ER chaperone BiP (18, 78). Moreover, our results are consistent with the observation that even the misfolded PiZ variant of AAT can escape the ER, albeit at much reduced levels (~15% of wild type) (27). Although a dominant effect of the PiZ variant on the export of wild-type AAT in heterozygous PiMZ individuals is not observed (27), this may simply be due to the efficient packaging of the wild-type protein through interaction with the COPII machinery even in the presence of a reduced level of vesicle carriers budding from the ER (reviewed in Ref. 79). This interpretation is consistent with the ability of the biosynthetic cargo selection machinery to efficiently sort transport-competent from transport-incompetent forms of VSV-G (80). Indeed, the defect leading to liver disease may be a consequence of loss of normal function of the AAT-specific selection machinery (81) in the absence of cargo, resulting in direct or indirect effects on the generation of COPII vesicles.

The observed effect of VSV-Gts on vesicle formation in vivo and in vitro demonstrates a more direct link between protein folding events and those involved in biosynthetic cargo selection than previously anticipated. Our results, combined with the observation that degradation of ER proteins is coupled to the translocation pore-component Sec61 (82), now suggest that protein import, folding, and export may be an integrated process. We anticipate that the effect of VSV-Gts on general budding at the restrictive temperature is at least partially exerted by non-productive interactions with Sar1 and Sec23/24. However, our ability to both prevent and enhance export by temperature shift demonstrates that such interactions are likely to be typical of the normal pathway involved in cargo selection (4). We propose that biosynthetic cargo such as VSV-G can therefore modulate COPII vesicle formation.

Is the observed ability of biosynthetic cargo accumulated in the ER to modulate vesicle formation a property that is shared at other export sites in the exocytic and endocytic pathways? In the case of clathrin, a similar regulation has been observed. Studies of trans-Golgi network (TGN) export sites have demonstrated that cargo molecules such as mannose 6-phosphate receptors and major histocompatibility complex II regulate the formation of AP1-driven clathrin-coated vesicles from the TGN in vivo (10-12). Although sorting is the primary function for vesicle budding from the TGN, the ER must coordinate cargo sorting and vesicle formation with quality control events associated with protein folding, oligomerization, and the degradation of misfolded cargo or cargo subunits that fail to be oligomerized into the mature protein (18, 64). We suggest that the detection and disposal of incompetent cargo by the degradative pathway may play an important role in eliminating potential dominant negative effects of such cargo on general ER export as we have observed for VSV-Gts and the AATPiZ variant. Indeed, evidence for the adverse effects of ER accumulation of cargo are found in the pathophysiology of disease. A subset of patients who express the PiZ variant of AAT develop liver injury (reviewed in Ref. 27). The partial effects of accumulated PiZ variant leading to a general decrease in budding raises the possibility that its accumulation may lead to liver injury by both decreasing the ability of the ER to efficiently export other critical biosynthetic cargo, in addition to possible negative effects related to induction of stress-related signaling responses (65). Related effects are evident in a number of neuronal and non-neuronal degenerative diseases that are also marked by accumulation of mutant cargo in the ER with severe consequences on cell growth (83, 84). Additional studies will be required to determine the general role of cargo in the development of these diseases.

It is now apparent that a variety of mechanisms may operate in the ER to sense, couple, and synchronize cargo processing, degradation, and export. We have raised the possibility that coupling between biosynthetic cargo and the COPII machinery may represent an important step in control of ER budding which will now need to be explored more fully.

    ACKNOWLEDGEMENT

The electron microscopy made extensive use of Core B supported by National Institutes of Health Grant CA 58689.

    FOOTNOTES

* This work was supported in part by Grants GM 42336 and CA 58689 from the National Institutes of Health (to W. E. B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to the memory of Thomas Kreis.

Dagger Recipient of a fellowship grant from the European Molecular Biology Organization and the Human Frontier Sciences Program.

§ Recipient of a Cystic Fibrosis Foundation Post-doctoral fellowship.

Recipient of a fellowship from the Human Frontier Sciences Program and the Muscular Dystrophy Association.

parallel To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, The Scripps Research Institute, 10666 N. Torrey Pines Rd., La Jolla, CA 92037.

The abbreviations used are: ER, endoplasmic reticulum; VSV-G, vesicular stomatitis glycoprotein; TGN, trans-Golgi network; VSV, vesicular stomatitis virus; GST, glutathione S-transferase; NRK, normal rat kidney; OKA, okadaic acid; CHX, cycloheximide; MSP, medium speed pellet; MSS, medium speed supernatant; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

2 M. Aridor and W. E. Balch, unpublished observations.

3 S. Bannykh and W. E. Balch, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
References

  1. Schekman, R., and Orci, L. (1996) Science 271, 1526-1533[Abstract]
  2. Aridor, M., and Balch, W. E. (1996) Trends Cell Biol. 6, 315-320[CrossRef]
  3. Hurtley, S. M., and Helenius, A. (1989) Annu. Rev. Cell Biol. 5, 277-307[CrossRef]
  4. Aridor, M., Weissman, J., Bannykh, S., Nouffer, C., and Balch, W. E. (1998) J. Cell Biol. 141, 61-70[Abstract/Free Full Text]
  5. Kuehn, M. J., Herrmann, M., and Schekman, R. (1998) Nature 391, 187-190[CrossRef][Medline] [Order article via Infotrieve]
  6. Schmid, S. L. (1997) Annu. Rev. Biochem. 66, 511-548[CrossRef][Medline] [Order article via Infotrieve]
  7. Bonifacino, J. S., Marks, M. S., Ohno, H., and Kirchhausen, T. (1996) Proc. Assoc. Am. Physicians 4, 285-295
  8. Iacopetta, B. J., Rothenberger, S., and Kühn, L. C. (1988) Cell 54, 485-489[Medline] [Order article via Infotrieve]
  9. Millar, K., Shipman, M., Trowbridge, I. S., and Hopkins, C. R. (1991) Cell 65, 621-632[Medline] [Order article via Infotrieve]
  10. Le Borgne, R., Griffiths, G., and Hoflack, B. (1996) J. Biol. Chem. 271, 2162-2170[Abstract/Free Full Text]
  11. Le Borgne, R., and Hoflack, B. (1997) J. Cell Biol. 137, 335-345[Abstract/Free Full Text]
  12. Salamero, J., Le Borgne, R., Saudrais, C., Goud, B., and Hoflack, B. (1996) J. Biol. Chem. 271, 30318-30321[Abstract/Free Full Text]
  13. Rothman, J. E. (1994) Nature 372, 55-63[CrossRef][Medline] [Order article via Infotrieve]
  14. Bannykh, S., Nishimura, N., and Balch, W. E. (1998) Trends Cell Biol. 8, 21-25[CrossRef][Medline] [Order article via Infotrieve]
  15. Hammond, C., and Helenius, A. (1994) Science 266, 456-458[Medline] [Order article via Infotrieve]
  16. De Silva, A. M., Balch, W. E., and Helenius, A. (1990) J. Cell Biol. 111, 857-866[Abstract]
  17. Braakman, I., Helenius, J., and Helenius, A. (1992) EMBO J. 11, 1717-1722[Abstract]
  18. Hammond, C., and Helenius, A. (1995) Curr. Biol. 7, 523-529
  19. Aridor, M., Bannykh, S. I., Rowe, T., and Balch, W. E. (1995) J. Cell Biol. 131, 875-893[Abstract]
  20. Balch, W. E., McCaffery, J. M., Plutner, H., and Farquhar, M. G. (1994) Cell 76, 841-852[Medline] [Order article via Infotrieve]
  21. Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534[Medline] [Order article via Infotrieve]
  22. Plutner, H., Davidson, H. W., Saraste, J., and Balch, W. E. (1992) J. Cell Biol. 119, 1097-1116[Abstract]
  23. Presley, J. F., Cole, N. B., Schroer, T. A., Hirschberg, K., Zaal, K. J. M., and Lippincott-Schwartz, J. (1997) Nature 389, 81-84[CrossRef][Medline] [Order article via Infotrieve]
  24. Bannykh, S. I., Rowe, T., and Balch, W. E. (1996) J. Cell Biol. 135, 19-35[Abstract]
  25. Rowe, T., Aridor, M., McCaffery, J. M., Plutner, H., and Balch, W. E. (1996) J. Cell Biol. 135, 895-911[Abstract]
  26. Sifers, R. N., Brashears-Macatee, S., Kidd, V. J., Muensch, H., and Woo, S. L. C. (1988) J. Biol. Chem. 263, 7330-7335[Abstract/Free Full Text]
  27. Sifers, R. N. (1995) Nat. Struct. Biol. 2, 355-357[Medline] [Order article via Infotrieve]
  28. Kreis, T. E. (1986) EMBO J. 5, 931-941[Abstract]
  29. Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007[Abstract/Free Full Text]
  30. Davidson, H. W., and Balch, W. E. (1993) J. Biol. Chem. 268, 4216-4226[Abstract/Free Full Text]
  31. Balch, W. E., and Rothman, J. E. (1985) Arch. Biochem. Biophys. 240, 413-425[Medline] [Order article via Infotrieve]
  32. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  33. Duden, R., Griffiths, G., Frank, R., Argos, P., and Kreis, T. E. (1991) Cell 64, 649-665[Medline] [Order article via Infotrieve]
  34. Pepperkok, R., Scheel, J., Horstmann, H., Hauri, H. P., Griffiths, G., and Kreis, T. E. (1993) Cell 74, 71-82[Medline] [Order article via Infotrieve]
  35. Palade, G. E. (1975) Science 189, 347-354[Medline] [Order article via Infotrieve]
  36. Davidson, H. W., McGowan, C. H., and Balch, W. E. (1992) J. Cell Biol. 116, 1343-1355[Abstract]
  37. Fabbri, M., Bannykh, S., and Balch, W. E. (1994) J. Biol. Chem. 269, 26848-26857[Abstract/Free Full Text]
  38. Pryde, J. G., Farmaki, T., and Lucocq, J. M. (1998) Mol. Cell. Biol. 18, 1125-1135[Abstract/Free Full Text]
  39. Saraste, J., and Kuismanen, E. (1984) Cell 38, 535-549[Medline] [Order article via Infotrieve]
  40. Saraste, J., and Svensson, K. (1991) J. Cell Sci. 100, 415-430[Abstract]
  41. Lafay, F. (1974) J. Virol. 14, 1220-1228[Medline] [Order article via Infotrieve]
  42. Storrie, B., Pepperkok, R., Stelzer, E. H., and Kreis, T. E. (1994) J. Cell Sci. 107, 1309-1319[Abstract/Free Full Text]
  43. Dunigan, D. D., and Lucas-Lenard, J. M. (1983) J. Virol. 45, 618-626[Medline] [Order article via Infotrieve]
  44. Le, A., Ferrell, G. A., Dishon, D. S., Le, Q.-Q. A., and Sifers, R. N. (1992) J. Biol. Chem. 267, 1072-1080[Abstract/Free Full Text]
  45. Wu, Y., Whitman, I., Molmenti, E., Moore, K., Hippenmeyer, P., and Perlmutter, D. H. (1994) Proc. Natl. Acad. Sci. U. S. A. 267, 1072-1080
  46. Kaiser, C. A., and Schekman, R. (1990) Cell 61, 723-733[Medline] [Order article via Infotrieve]
  47. Keller, G.-A., Glass, C., Louvard, D., Steinberg, D., and Singer, S. J. (1986) J. Histochem. Cytochem. 34, 12223-1230
  48. Mizuno, M., and Singer, S. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5732-5736[Abstract]
  49. Lodish, H. F., Kong, N., Snider, M., and Strous, G. J. (1983) Nature 304, 80-83[Medline] [Order article via Infotrieve]
  50. Lodish, H. F. (1988) J. Biol. Chem. 263, 2107-2110[Free Full Text]
  51. Le, A., Steiner, J. L., Ferrell, G. A., Shaker, J. C., and Sifers, R. N. (1994) J. Biol. Chem. 269, 7514-7519[Abstract/Free Full Text]
  52. Tisdale, E. J., Plutner, H., Matteson, J., and Balch, W. E. (1997) J. Cell Biol. 137, 581-593[Abstract/Free Full Text]
  53. Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M. F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994) Cell 77, 895-907[Medline] [Order article via Infotrieve]
  54. Kreis, T. E., Lowe, M., and Pepperkok, R. (1995) Annu. Rev. Cell Dev. Biol. 11, 677-706[CrossRef][Medline] [Order article via Infotrieve]
  55. Schekman, R., Barlowe, C., Bednarek, S., Campbell, J., Doering, T. D., Duden, R., Kuehn, M., Rexach, M., Yeung, T., and Orci, L. (1995) Cold Spring Harbor Symp. Quant. Biol. 60, 11-21[Medline] [Order article via Infotrieve]
  56. Rothman, J. E., and Wieland, F. (1996) Science 272, 272-234
  57. Helms, J. B., Palmer, D. J., and Rothman, J. E. (1993) J. Cell Biol. 121, 751-760[Abstract]
  58. Malhotra, V., Serafini, T., Orci, L., Shepherd, J. C., and Rothman, J. E. (1989) Cell 58, 329-336[Medline] [Order article via Infotrieve]
  59. Melancon, P., Glick, B. S., Malhotra, V., Weidman, P. J., Serafini, T., Orci, L., and Rothman, J. E. (1989) Soc. Gen. Physiol. Ser. 44, 175-188[Medline] [Order article via Infotrieve]
  60. Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J. B., and Rothman, J. E. (1993) J. Cell Biol. 123, 1365-1371[Abstract]
  61. Stamnes, M. A., and Rothman, J. E. (1993) Cell 73, 999-1005[Medline] [Order article via Infotrieve]
  62. Ostermann, J., Orci, L., Tani, K., Amherdt, M., Ravazzola, M., Elazar, Z., and Rothman, J. E. (1993) Cell 75, 1015-1025[Medline] [Order article via Infotrieve]
  63. Dominguez, M., Dejgaard, K., Fullekrug, J., Sahan, S., Fazel, A., Paccaud, J.-P., Thomas, D. Y., Bergeron, J. J. M., and Nilsson, T. (1998) J. Cell Biol. 140, 751-765[Abstract/Free Full Text]
  64. Brodsky, J. L., and McCracken, A. A. (1997) Trends Cell Biol. 7, 151-156[CrossRef]
  65. Pahl, H. L., and Baeuerle, P. A. (1997) Trends Cell Biol. 7, 50-55[CrossRef]
  66. Itin, C., Roche, A.-C., Monsigny, M., and Hauri, H.-P. (1996) Mol. Biol. Cell 7, 483-493[Abstract]
  67. Nichols, W. C., Seligsohn, U., Zivelin, A., Terry, V. H., Hertel, C. E., Wheatley, M. A., Moussalli, M. J., Hauri, H.-P., Ciavarella, N., Kaufman, R. J., and Ginburg, D. (1998) Cell 93, 61-70[Medline] [Order article via Infotrieve]
  68. Vollenweider, F., Kappeler, F., Itin, C., and Hauri, H.-P. (1998) J. Cell Biol. 142, 377-389[Abstract/Free Full Text]
  69. Yeung, T., Barlowe, C., and Schekman, R. (1995) J. Biol. Chem. 270, 30567-30570[Abstract/Free Full Text]
  70. Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S. Y., Hamamoto, S., Schekman, R., and Yeung, T. (1998) Cell 93, 263-275[Medline] [Order article via Infotrieve]
  71. Springer, S., and Schekman, R. (1998) Science 281, 698-700[Abstract/Free Full Text]
  72. Stone, S., Sacher, M., Mao, Y., Carr, C., Lyons, P., Quinn, A. M., and Ferro-Novick, S. (1997) Mol. Biol. Cell 8, 1175-1181[Abstract]
  73. Lian, J. P., and Ferro-Novick, S. (1993) Cell 73, 735-745[Medline] [Order article via Infotrieve]
  74. Taylor, R. S., Jones, S. M., Nordeen, M. H., and Howell, K. E. (1997) Mol. Biol. Cell 8, 1911-1931[Abstract/Free Full Text]
  75. Orci, L., Stamnes, M., Ravazzola, M., Amherdt, M., Perrelet, A., Sollner, T. H., and Rothman, J. E. (1997) Cell 90, 335-349[Medline] [Order article via Infotrieve]
  76. Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316[Abstract/Free Full Text]
  77. Lewis, M. J., and Pelham, H. R. B. (1992) Cell 68, 353-364[Medline] [Order article via Infotrieve]
  78. Hammond, C., and Helenius, A. (1994) J. Cell Biol. 126, 41-52[Abstract]
  79. Sifers, R. N., Finedgold, M. J., and Woo, S. L. C. (1992) Semin. Liver Dis. 12, 301-310[Medline] [Order article via Infotrieve]
  80. Zagouras, P., Ruusala, A., and Rose, J. K. (1991) J. Virol. 65, 1976-1984[Medline] [Order article via Infotrieve]
  81. Sifers, R. N., Rogers, B. B., Hawkins, H. K., Finegold, M. J., and Woo, S. L. C. (1989) J. Biol. Chem. 264, 15696-15700[Abstract/Free Full Text]
  82. Wiertz, E. J. H. J., Tortorella, D., Bogyo, M., Yu, J., Mothes, W., Jones, T. R., Rapoport, T. A., and Ploegh, H. L. (1996) Nature 384, 432-438[CrossRef][Medline] [Order article via Infotrieve]
  83. Brooks, D. A. (1997) FEBS Lett. 409, 115-120[CrossRef][Medline] [Order article via Infotrieve]
  84. Amara, J. F., Cheng, S. H., and Smith, A. E. (1992) Trends Cell Biol. 2, 145-149[CrossRef]
  85. Rowe, T., and Balch, W. E. (1995) Methods Enzymol. 257, 49-53[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.