Purification of Sec23–24 from Rat Liver Cytosol
To analyze the role of COPII in cargo selection and ER export, we first purified the mammalian Sec23–24 complex present in rat liver cytosol to homogeneity. Using a mouse clone of mammalian Sec23 (refer to Materials and Methods), we prepared antipeptide and antiprotein antibodies to Sec23. The mouse clone is 99% homologous to the previously reported human clone of Sec23a (Paccaud et al., 1996
). Rabbit polyclonal antibodies recognized an ~85-kD protein in the cytosol using Western blotting that is also recognized by an antibody generated against yeast Sec23 (Hicke and Schekman, 1989
; Hicke et al., 1992
), which has been used to morphologically localize Sec23 to the ER in intact pancreatic acinar cells (Orci et al., 1991
). Our polyclonal Sec23 antibody immunoprecipitated an ~85-kD protein complexed with a 120-kD protein from rat liver cytosol (data not shown).
The Sec23–24 complex was purified from rat liver cytosol through sequential steps involving ammonium sulfate precipitation, S-300 gel filtration, DEAE–ion exchange, and HAP chomatography (Fig. ) (refer to Materials and Methods) based on Western blotting using the Sec23 specific antibody. The purified Sec23–24 complex (Fig. C
, lane e
) contained two major bands of 85 and 120 kD. The identity of the 85-kD band was established by microsequencing and found to be identical to the human homologue (Paccaud et al., 1996
; data not shown). The 120-kD band was identified as the mammalian homologue of yeast Sec24 by microsequencing (data not shown). The Sec24 component was found to particularly labile to partial proteolysis as observed in yeast (Yeung et al., 1995
), accounting for faster migrating minor bands in the purified preparation (Fig. C
, lane e
) as detected by Western blotting using specific antibody to Sec24 (data not shown).
Figure 1 Purification of Sec23–24 from rat liver cytosol. The Sec23–24 complex was purified from rat liver cytosol as described in the Materials and Methods. A and B illustrate typical elution profiles from the DEAE and HAP chromatography steps, (more ...)
Yeast Sec23 was previously reported to be a Sar1-specific GTPase-activating protein (GAP) (Yoshihisa et al., 1993
), accelerating GTP hydrolysis ~15-fold over the intrinsic rate of hydrolysis. To address the functionality of the purified complex as a Sar1 GAP, we tested the GTP hydrolysis activity in the presence or absence of the purified complex. The purified mammalian Sec23–24 complex accelerated GTP hydrolysis of mammalian Sar1 up to 100-fold the intrinsic rate (Fig. ), demonstrating its role as a Sar1 GAP. As a control, the complex itself did not show any GTPase activity (data not shown). A similar result was obtained with GST-tagged Sec23, showing that the Sec23 component of the complex contained GAP activity, as reported previously for the yeast complex (Yoshihisa et al., 1993
Figure 2 Sec23 and the Sec23–24 are functional GAP proteins for the mammalian Sar1 protein. Recombinant Sar1 was incubated in the presence or absence of GST– Sec23 or purified Sec23–24 and then GAP activity was monitored as described (more ...)
Sec23 Is the Minimal Cytosolic COPII Component That Can Be Recruited to Membranes in Response to Sar1 Activation
To begin to identify the possible protein interactions initiating cargo sorting into COPII vesicles, we examined the recruitment of purified Sec23–24 to ER membranes. Incubation of microsomes with rat liver cytosol led to a temperature- and Sar1-dependent recruitment of the Sec23 component to membranes when measured by pelleting of total membranes (Fig. A
). This result is consistent with our previous observation that in semi-intact cells, after activation of the Sar1 GTPase, COPII is recruited specifically to the ER and can be colocalized with the mobilized VSV-G based on indirect immunofluorescence (Aridor et al., 1995
Figure 3 Sec 23 represents the minimal coat component that is recruited to the membranes by the Sar1 GTPase. (A) Microsomes were incubated with rat liver cytosol for 10 min on ice or at 32°C in the absence or presence of 1 μg of activated (more ...)
Stable recruitment of COPII from cytosol to membranes was observed only in the presence of an ATP regenerating system, micromolar concentrations of GTP and the Sar1–GTP–restricted mutant to prevent coat disassembly (Fig. A
). The ATP regenerating system could not be replaced by inclusion of the nonhydrolyzable analogue of ATP, ATPγS (data not shown). The requirement for the ATP regenerating system was still observed when recruitment was initiated in the absence of Sar1–GTP, but in the presence of the nonhydrolyzable analogue of GTP, GTPγS, activated to the endogenous cytosolic Sar1 protein (Fig. B
). COPII recruitment required membrane-associated components as limited proteolysis of the membranes prevented binding (data not shown). No recruitment was observed when excess GTP was added together with GTPγS in the absence of ATP (Fig. B
) and excess GTP significantly reduced GTPγS-induced recruitment in the presence of ATP (Fig. B
). GTPγS is required, therefore, to stabilize endogenous Sar1 in its active state to detect COPII binding to membranes. In contrast, addition of millimolar concentrations of GTP with the Sar1–GTP mutant in the absence of the ATP-regenerating system efficiently supported recruitment (Fig. B
). Some of the GTP may be used to generate trace levels of ATP, or added hydrolyzable nucleotide may be used by a protein that does not differentiate between ATP and GTP such as casein kinase II. Interestingly, casein kinase II–like enyzmes have been implicated in coat recruitment (Bonifacino et al., 1996
). In any case, these results suggest that Sec23 recruitment involves an additional ATP/GTP-dependent function that is dependent on hydrolysis and is distinct from that of Sar1. A similar requirement has been found for the ARF1-dependent recruitment of AP1 and AP3 adaptor complexes involved in clathrin-coated vesicle formation (Traub et al., 1993
; Simpson et al., 1996
To define the minimal cytosolic components that could be recruited to membranes in the presence of activated Sar1, we examined the recruitment of purified Sec23–24 complex from rat liver cytosol. As observed for recruitment from cytosol, recruitment of purified Sec23–24 was temperature, ATP, and Sar1–GTP dependent (Fig. , compare A and C, a–d). Moreover, identical results were also observed when recombinant His6-tagged–Sec23 monomer was used in the absence of the Sec24 protein (Fig. C, lanes e–h). Thus, the ATP/GTP requirement for Sec23 recruitment is due to an activity present on membranes, and Sec23 represents the minimal cytosolic component of the COPII machinery that can be stably recruited to microsomal membranes after activation of the Sar1 GTPase.
VSV-G Interacts with the COPII Machinery in a Sar1-dependent Manner
To address the role of COPII components in cargo selection and vesicle budding, we used an assay that reconstitutes the release of the type 1 transmembrane protein VSV-G into COPII–coated vesicles from mammalian microsomes (Rowe et al., 1996
). To follow vesicle budding in vitro, a postnuclear supernatant fraction is prepared from homogenates of cells containing VSV-G in the ER. Membranes are incubated in a transport cocktail containing cytosol from rat liver and an energy source in the form of ATP. The fraction of VSV-G 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 (16,000 g
) pellet (MSP) from slowly sedimenting ER-derived vesicles that are released into the medium speed supernatant (MSS). Carrier vesicles present in the MSS are subsequently recovered in a high speed (100,000 g
) pellet (HSP), and the amounts of VSV-G in the MSP or HSP fractions are measured by SDS-PAGE and quantitative Western blotting.
To define the minimal components required for vesicle budding, membranes were washed with high salt to remove any residual bound Sar1, Sec23–24, and Sec13–31 complex (Barlowe et al., 1994
; data not shown). These membranes remained export competent, since incubation with cytosol and Sar1–GTP led to the accumulation of VSV-G–containing vesicles (Fig. A
). Budding was inhibited by incubation with the Sar1A-GDP–restricted mutant (Fig. A
), a result consistent with our previous demonstration that VSV-G export from the ER is regulated by the Sar1 GTPase (Rowe et al., 1996
). High salt-washed membranes retained their ability to recruit a GST-tagged Sec23 (GST–Sec23) monomer in a Sar1A-dependent manner (Fig. B
). However, addition of activated Sar1A and GST–Sec23 did not support COPII vesicle formation (Fig. A
). These results indicate that GST–Sec23, in the absence of Sec24 and Sec13–31, is not sufficient for budding.
Figure 4 VSV-G can be detected in a complex with Sec23. (A) Salt-washed microsomes prepared as described in Materials and Methods were incubated at 32°C for 30 min in the presence of rat liver cytosol (a and b) or GST–Sec23 (c) in the presence (more ...)
Having defined conditions that do not support vesicle budding, yet support stable recruitment of the GST–Sec23 monomer to membranes, we analyzed whether cargo becomes associated with the recruited Sec23 containing a partial coat. For this purpose, salt-washed microsomes incubated with recombinant GST–Sec23 and/or Sar1A mutants were pelleted, washed, and then solubilized in a detergent containing buffer. After centrifugation to remove insoluble material, the supernatant was incubated with GS beads and then bound GST–Sec23 was quantitated using Western blotting. GST–Sec23 recovery on GS beads was both Sar1A and temperature dependent (Fig. B, b and c). Incubation in the presence of the activated Sar1–GTP– restricted mutant (to prevent coat disassembly) resulted in an ~100-fold increase in GS bead-bound Sec23 (Fig. B, d). Strikingly, VSV-G was also recovered on GS beads and its recruitment directly mirrored the requirements for GST–Sec23 binding (Fig. B, top). On the average, 15– 20% of the total soluble VSV-G was recovered on GS beads (Fig. C, top, compare a and b), a value comparable to the amount of VSV-G generally released into vesicles in the presence of cytosol and ATP (Fig. A, a). Identical results were observed in a converse set of experiments in which an antibody that recognizes the lumenal domain of VSV-G was used in place of GS beads to immunoprecipitate the VSV-G/GST–Sec23–containing complex (data not shown). Recruitment was selective as ribophorin II and calnexin, abundant ER marker proteins, could not be detected in the protein complexes bound to GS beads (Fig. C, bottom, compare a and b). The soluble chaperone BIP was also excluded from the complex (data not shown). Thus, Sec23 is sufficient to initiate VSV-G recruitment in concert with Sar1A activation, demonstrating a role for Sec23 in cargo sorting that is initiated before vesicle budding.
Sar1 and the Sec23–24 Complex Are Both Required for the Formation of a Cargo Containing Prebudding Intermediate
Although the addition of GST–Sec23 in the absence of the Sec24 component promoted the selective recruitment of cargo, it did not support further vesicle formation. To use the purified Sec23–24 for analysis of cargo recruitment and vesicle formation and to analyze whether Sar1 is a component of the prebudding complex, we modified the activated form of Sar1, Sar1–GTP, with GST to generate a GST/Sar1–GTP chimera (GST–Sar1–GTP). We tested whether the GST–Sar1–GTP chimera would promote the formation of VSV-G–containing vesicles. We took advantage of the fact that after salt wash, Sar1 becomes a limiting component of the budding reaction. This requirement could either be supplemented with excess cytosol (data not shown) or with added exogenous recombinant Sar1 (Fig. , c). Washed microsomes were resuspended in a transport reaction mix in the presence of ATP, cytosol, and various Sar1 recombinant proteins for 30 min and then the release of VSV-G to the HSP was measured. Supplementing the assay with either wild-type recombinant Sar1 (Fig. A, compare b to c), Sar1–GTP (Fig. A, compare b to d), or GST–Sar1–GTP (Fig. A, compare b to e) supported efficient vesicle formation.
Figure 5 GST-tagged Sar1–GTP supports vesicle budding from salt-washed microsomal membranes. (A) Salt-washed microsomes prepared as described in Materials and Methods were incubated in a budding reaction with cytosol on ice (a) or for 30 min at 32°C (more ...)
To determine if GST–Sar1–GTP protein was incorporated into VSV-G–containing vesicles, microsomes were incubated in the presence of cytosol and GST–Sar1–GTP for 30 min and then ER-derived vesicles were released into the immunoisolated HSP using magnetic beads coated with anti–VSV-G cytoplasmic tail antibody (P504), an approach we have previously used to characterize both morphologically and biochemically the composition of Sec23-containing COPII vesicles generated in vitro (Rowe et al., 1996
). Like Sec23, GST–Sar1–GTP could be readily detected on affinity-purified vesicles using Western blotting with an antibody specific to Sar1 based on its higher molecular weight (47 kD) compared to wild-type Sar1 (25 kD) (Fig. B
Because GST–Sar1–GTP is active in both coat recruitment and vesicle formation, we examined the ability of this chimera to isolate the VSV-G–containing cargo complex in the presence or absence of Sec23–24 purified from rat liver cytosol. We found that GST–Sar1–GTP promoted a temperature-dependent recruitment of Sec23–24 to microsomal membranes as was observed for GST–Sec23 (refer to Fig. A) (data not shown). Under these conditions, the recruited components did not support vesicle budding in the absence of Sec13–31 (see below and Fig. ). After incubation, the membranes were solubilized and then the detergent-soluble fraction was incubated with GS beads. Proteins bound to GS beads were subjected to immunoblot and protein analysis. As shown in Fig. , incubation of the membranes in the presence of Sec23–24 but in the absence of the GST–Sar1–GTP did not promote cargo binding to GS beads (Fig. A, a). Similarly, addition of GST– Sar1–GTP alone only led to the recovery of a small amount (<1%) of total VSV-G (Fig. A, b), presumably reflecting a residual contamination of salt-washed membranes with COPII components. However, addition of both Sec23–24 and GST–Sar1–GTP led to efficient VSV-G recruitment (Fig. A, c). VSV-G recruitment was temperature dependent (data not shown) and saturable in the presence of increasing Sec23–24 (Fig. D) as further addition of the complex had no effect on the amount of VSV-G recruited to beads or on VSV-G released in the budding assay in a number of experiments (data not shown). The amount of VSV-G recovered under optimal conditions was comparable to that observed by incubation in the presence of GST–Sec23 and Sar1–GTP (refer to Fig. ). Strikingly, recruitment was selective and excluded ER resident proteins such as ribophorin II (Fig. B) or calnexin (data not shown).
Figure 7 Vesicle budding from the ER requires Sar1, Sec23–24, and Sec13–31. (A) The mammalian Sec13–31 complex was partially purified from rat liver cytosol as described in the Materials and Methods. The presence of Sar1, Sec23, (more ...)
Figure 6 VSV-G can be isolated in a complex with a GST-tagged Sar1–GTP and the mammalian Sec23–24 complex. (A–C) Salt-washed microsomes were incubated at 32°C for 30 min in the presence of Sec23–24 complex (lanes a, d (more ...)
Silver staining of the immunoisolated complex demonstrated the efficient recruitment of Sec23–24 (Fig. C, arrows). Although a number of proteins were bound to GS beads in the presence of Sec23–24 alone (Fig. C, g), a control condition that does not support either cargo recruitment or vesicle budding, illustrating nonspecific background binding to GS beads, incubation in the presence of GST–Sar1–GTP alone (Fig. C, h) or in combination with Sec23–24 (Fig. C, i), led to the recruitment of only a limited number of proteins above the background (Fig. C, gray arrowheads). The identity and possible role of these proteins in COPII–mediated cargo selection and vesicle formation are currently under investigation. Importantly, a protein band corresponding to VSV-G based on immunoblotting (Fig. , A and C, compare b with i, large arrowhead) was markedly enhanced in the presence of GST– Sar1–GTP and Sec23–24, but not in incubations lacking either of the components. These results directly demonstrate the ability of GST–Sar1 to recruit VSV-G into a cargo-containing complex. Interestingly, the level of recruitment of VSV-G was similar to that of components bound to the complex during initiation of vesicle budding by the addition of Sar1 alone (Fig. C, gray arrowheads), suggesting that a limiting component related to Sar1 recruitment is involved in cargo selection.
The Prebudding Complex Is an Intermediate in ER-to-Golgi Transport
The ability to recruit VSV-G to a detergent-soluble complex containing GST–Sar1 and Sec23–24 led us to examine whether this complex is an intermediate in vesicle budding. For this purpose, a partially purified Sec13–31 complex was prepared from rat liver cytosol. As shown in Fig. A, this fraction lacks Sar1, Sec23, and Sec24, but is substantially enriched (nearly 1,000-fold based on Western blotting) in the Sec13 component. When each of the components was incubated separately with VSV-G containing ER microsomes, vesicle budding was not observed (Fig. B). However, incubation of Sar1–GTP or GST–Sar1–GTP in the presence of both Sec23–24 and Sec13–31 led to efficient recovery of VSV-G in COPII coated vesicles.
To verify the above requirements for Sar1, the Sec23–24 and the Sec13–31 complexes for the appearance of VSV-G–containing vesicles, we examined the effects of each of these components on vesicle budding in vitro using immunoelectron microscopy. Semi-intact cells, a population of cells in which the plasma membrane is selectively perforated (Beckers et al., 1987
; Plutner et al., 1992
), faithfully reconstitute cargo selection (Balch et al., 1994
) and transport of VSV-G from the ER to Golgi compartments (Davidson and Balch, 1993
; Plutner et al., 1991
; Nuoffer et al., 1994
; Peter et al., 1994
; Tisdale et al., 1997
). Incubation in the presence of cytosol leads to the sorting and concentration of VSV-G in ER-derived buds and pre-Golgi intermediates (Fig. A
) as demonstrated previously using morphometry (Balch et al., 1994
; Pind et al., 1994
). An identical result was observed in the presence of the COPII components Sar1–GTP, Sec23–24, and Sec13–31 (Fig. B
). Typically, VSV-G was concentrated ~5–15-fold in both ER-associated buds as well as in free vesicles over that observed in the ER membrane. Interestingly, in these incubations containing purified COPII components, we often found VSV-G concentrated on the face of the ER adjacent to regions of high budding activity (Fig. B
, top right
), suggesting the budding may be limiting, relative to cargo selection and concentration under these conditions. Since these incubation conditions lack COPI components required for retrograde recycling from pre-Golgi intermediates (Aridor et al., 1995
; Rowe et al., 1996
) or additional cytosolic factors required for transport to the Golgi (Balch, W.E., unpublished data), the observed concentration of VSV-G in vesicles using only COPII components provides an additional line of evidence that sorting and concentration occurs during cargo selection from the ER (Balch et al., 1994
; Bannykh et al., 1996
; Rowe et al., 1996
Figure 8 Immunolocalization of VSV-G in semi-intact cells incubated in the presence of cytosol or purified COP II components. Incubation of semiintact cells in the presence of ATP and the indicated components and localization of VSV-G using immunoelectron microscopy (more ...)
In contrast to our ability to readily detect VSV-G in vesicles when semi-intact cells were incubated in the presence of cytosol or the presence of COPII components, VSV-G–containing vesicles were not detected when cells were incubated in the presence of Sar1–GTP alone, consistent with the inability of Sar1–GTP to support budding from ER microsomes in vitro. However, incubation of Sar1 and the Sec23–24 complex together led to a population of structures that appeared as either partial buds or regions of the ER membrane where VSV-G was concentrated in patches relative to its general diffuse distribution before incubation in vitro (Fig. C). These morphological results support our supposition from biochemical studies that Sar1– GTP and the Sec23–24 complex are sufficient to promote an interaction with VSV-G and that all of the components are necessary to direct budding from the ER.