A Putative Heterotrimeric G Protein Inhibits the Fusion of COPI-coated Vesicles
SEGREGATION OF HETEROTRIMERIC G PROTEINS FROM COPI-COATED VESICLES*

J. Bernd HelmsDagger §, Désiré Helms-BronsDagger , Britta BrüggerDagger , Ioannis GkantiragasDagger , Heike EberleDagger , Walter NickelDagger , Bernd Nürnberg, Hans-Hermann Gerdesparallel , and Felix T. WielandDagger

From the Dagger  Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg,  Freie Universitaet Berlin, Universitätsklinikum Rudolf Virchow, Institut für Pharmakologie, Thielallee 67-73, 14195 Berlin, and parallel  Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Heterotrimeric G proteins have been implicated in the regulation of intracellular protein transport, but their mechanism of action remains unclear. In vivo, secretion of chromogranin B, tagged with the green fluorescent protein, was inhibited by the addition of a general activator of trimeric G proteins (AlF4-) to stably transfected Vero cells and resulted in an accumulation of the tagged protein in the Golgi apparatus. In an in vitro assay that reconstitutes intra-Golgi protein transport, we find that a membrane-bound and AlF4--sensitive factor is involved in the fusion reaction. To determine whether this effect is mediated by a heterotrimeric G protein localized to COPI-coated transport vesicles, we determined the presence of G proteins on these vesicles and found that they were segregated relative to the donor membranes. Because G proteins do not have an obvious sorting, retention, or retrieval signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of protein secretion mediated by heterotrimeric G proteins.

    INTRODUCTION
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Introduction
Procedures
Results
Discussion
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Vesicular protein transport between intracellular compartments involves the budding, targeting, and fusion of transport vesicles. These highly specific events are controlled by proteins that belong to a general protein machinery (1). Among these are various small molecular weight GTP-binding proteins as well as heterotrimeric GTP-binding (G)1 proteins that are known to regulate vesicular transport. Of the small molecular weight GTP-binding proteins, the ADP-ribosylation factor (ARF) is a stoichiometric component of COP-coated vesicles (2) and is involved in coatomer recruitment (3, 4) as well as in uncoating of COP-coated vesicles (5). Another member of the small molecular weight GTP-binding proteins, the Rab-family of proteins, is thought to act in concert with v- and t-SNAREs, which regulate the specificity of membrane fusion (6).

The involvement of heterotrimeric G proteins in intracellular protein transport was initially suggested by the finding that AlF4-, an activator of heterotrimeric but not of monomeric G proteins (7), inhibits intra-Golgi transport in a cell-free system (8). In vivo evidence for the involvement of heterotrimeric G proteins comes from overexpression of Galpha i3, which localizes to the Golgi complex and inhibits protein transport through the secretory pathway (9).

The mechanisms of the involvement of G proteins in intracellular protein traffic remains to be established. Probes that interfere with the classical G protein cycle at the plasma membrane have been used to study a G protein cycle in reconstituted intracellular vesicular transport assays, but conflicting results have been obtained (reviewed in Ref. 10). There seems to be a possibility of alternative G protein cycles at the early stages of the secretory pathway, whereas a classical G protein cycle may operate at later stages (10). Alternative G protein cycles may include interactions with ARF protein, either via a direct interaction of Gbeta gamma with ARF (reviewed in Ref. 10) or via interaction with an ARF GTPase-activating protein (11). Recently, G proteins have been implicated in the maintenance of the Golgi structure (12, 13). It is not clear, however, whether this signaling pathway is the same as the one involved in protein secretion.

To investigate the role of G proteins in early stages of the secretory pathway, we expanded initial experiments that showed an inhibitory effect of AlF4- in intra-Golgi transport (8). We show that the action of AlF4- is distinct from the action of GTPgamma S. Heterotrimeric G proteins are excluded from COPI-coated transport vesicles, possibly by interaction with other proteins in a detergent-insoluble complex.

    EXPERIMENTAL PROCEDURES
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Materials-- L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin was obtained from Worthington Biochemical Corp., Freehold, NJ. Soybean trypsin inhibitor was purchased from Sigma. CHO Golgi membranes were isolated as described (14). Bovine brain cytosol was prepared according to Malhotra et al. (15). A total membrane fraction from normal rat kidney cells was prepared by passing a 2-ml cell pellet, resuspended in a total volume of 15 ml of 20 mM Tris/HCl, pH 7.4, 1 mM EDTA, 50 mM NaCl (TEN buffer) several times through a G25 needle until more than 90% of the cells were broken as judged by trypan blue permeability. Whole cells and nuclei were spun down by centrifugation for 5 min at 500 × g. The postnuclear supernatant was centrifuged 45 min at 100,000× g, and the membrane pellet was resuspended in 8 ml of TEN buffer and stored at - 80 °C. Antibodies used against the G protein-specific subunits are AS 6 (Galpha oc), AS 232 (Galpha 12), AS 266 (Galpha ic), AS 348 (Galpha s), AS 369 (Galpha q/11) and are described in (16). AS398 (Gbeta c) is described in (17). In some experiments a Galpha i3- and a Galpha i1/2-specific antibody from NEN Life Science Products was used (EC/2 and AS/7, respectively). Polyclonal rabbit antibodies against p23 (#1327) and beta '-COP (#891) were used as described (18, 19). AlF4- was freshly prepared as a 10× stock solution by premixing equal volumes of 1 mM AlCl3 with 600 mM NaF.

Cell-free Intra-Golgi Transport Assay-- The standard incubation conditions were as described previously (14, 15).

In Vivo Secretion of Green Fluorescent Protein-tagged Human Chromogranin B (hCgB-GFP)-- Vero cells (clone V7) stably transfected with a hCgB-GFP fusion protein were grown on glass coverslips to ~40% confluency, and expression of the GFP construct was as described (20). Cells were mounted in Fluoromount G (Biozol) and analyzed using a Zeiss Axiovert 35 microscope equipped with the appropriate filters for fluorescein isothiocyanate-derived fluorescence.

Protease Digest-- 10 µg of isolated CHO Golgi membranes was pre-incubated in 25 mM Hepes/KOH, pH 7.2, 20 mM KCl, 2.5 mM magnesium acetate, and 10 µM GDP to analyze the G protein in its inactive conformation, which is more susceptible to protease digest (21). The membranes were then incubated with 2.5 µg of trypsin and/or 25 µg of soybean trypsin inhibitor for 15 min at 30 °C. The Golgi membranes were pelleted through a sucrose cushion (15% sucrose w/v) at 14,000 × g for 30 min. The proteins were analyzed by SDS-PAGE and subsequent Western blot analysis (ECL) using a Galpha i3-specific antibody (EC/2) and a p23-specific antibody raised against a luminal part of the protein (#1327) (18). Protein bands were quantified by densitometry measurements using a flatbed scanner and NIH image software (National Institutes of Health, U. S. A.).

Isolation of COPI-coated Vesicles-- COPI-coated vesicles were isolated from large scale incubation of CHO Golgi membranes with bovine brain cytosol under standard cell free assay conditions in the presence of 20 µM GTPgamma S or 20 µM GTPgamma S + AlF4- (50 µM AlCl3 + 30 mM NaF) (15, 22).

Lipid Analysis-- Lipids were extracted according to Bligh and Dyer (23) in the presence of 14:0/14:0 phosphatidylcholine (PC) as a nonnatural standard for quantitation. Lipids were analyzed and quantified by nano-electrospray ionization tandem mass spectrometry (24). PC was measured by detection of (M + H)+ ions by precursor scanning for m/z 184 (collision cell offset, -35 V).

Isolation of a Detergent-insoluble Fraction from Golgi Membranes-- Golgi membranes (5 µg) were pelleted at 14,000 × × g at 4 °C for 30 min and resuspended in 50 µl of 25 mM Pipes, pH 6.5, 2 mM EDTA, 150 mM NaCl (PEN buffer, or as described in the legends) containing 1% detergent, as indicated in the figure legend. After a 30-min incubation on ice, the detergent-insoluble fraction was isolated by centrifugation at 14,000 × g at 4 °C for 30 min. The pellet was resuspended in SDS-PAGE sample mixture and analyzed by SDS-PAGE and subsequent Western blot analysis (ECL) using antibodies as indicated in the figure legends.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

In Vivo Inhibition of Secretion by an AlF4--sensitive Factor-- Heterotrimeric G proteins have been localized to intracellular membranes along the secretory pathway including the endoplasmic reticulum and the Golgi complex (reviewed in Ref. 10). To determine at what stage G proteins might be involved in the regulation of intracellular protein transport, we used hCgB-GFP (20, 25), a marker of the secretory pathway. The fusion protein was stably transfected in Vero cells and is expressed upon induction with butyrate (20). hCgB-GFP is secreted from the cells at 37 °C, resulting in the absence of a fluorescent signal in these cells as determined by fluorescent microscopy (Fig. 1, bottom left panel). When, however, AlF4- is added to these cells at 37 °C, a perinuclear staining is observed (Fig. 1, bottom right panel). This indicates that the secretory pathway is inhibited at the stage of the fluorescent signal. A similar fluorescent signal was observed when cells were incubated at 15 and 20 °C, conditions known to block protein transport through the Golgi complex or at the trans-Golgi network (Fig. 1, top panels) and which is in agreement with previous experiments (20, 25). These data indicate that the hCgB-GFP accumulates in the Golgi complex upon treatment of the cells with AlF4-. No inhibition of protein export from the endoplasmic reticulum is observed. This is a surprising observation as G proteins have been implicated in the export of proteins from the endoplasmic reticulum (26). Alternatively, trimeric G proteins at the Golgi complex might be more intimately involved in regulation of protein secretion than at the endoplasmic reticulum.


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Fig. 1.   AlF4- inhibits anterograde transport through the Golgi of a hCgB-GFP fusion protein. Vero cells stably transfected with a hCgB fusion protein were incubated in the presence of 2 mM butyrate to stimulate protein expression. After 20 h at 37 °C, cells were incubated with AlF4- for 1 h at 37 °C, incubated at 15 °C for 1 h, incubated at 20 °C for 1 h, or incubated at 37 °C for 1 h as a control. Cells were fixed, permeabilized, and analyzed as described under "Experimental Procedures."

Inhibition of COPI-mediated Transport by an AlF4--sensitive Factor-- To further define an AlF4--sensitive factor responsible for inhibition of protein transport through the Golgi, we employed a cell-free system that reconstitutes COPI-coated vesicular transport between Golgi cisternae (14, 15). This assay previously revealed the involvement of a GTPgamma S- and an AlF4--sensitive factor (8). In subsequent publications, the GTPgamma S-sensitive factor was identified as a member(s) of the ARF family (2, 27). Here, we determined whether inhibition of COPI-mediated transport by AlF4- is dependent on the cytosol (high speed supernatant) concentration. In agreement with previous studies, COPI-mediated transport of the VSV G protein is dependent on the presence of cytosolic factors (14), and only at high concentrations of cytosol, GTPgamma S inhibits transport of the VSV G protein (Fig. 2 and Ref. 8). This indicates that the GTPgamma S-sensitive factor is recruited from the cytosol (8). In contrast, inhibition of transport by AlF4- does not show any cytosol dependence, indicating that this factor resides on the Golgi membranes (Fig. 2). This is in agreement with the membranous localization of intracellular heterotrimeric G proteins (28), although it cannot be excluded that AlF4- acts on some other proteins as well (see "Discussion").


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Fig. 2.   The AlF4--sensitive factor resides on Golgi membranes. Transport assays were performed under standard conditions (see "Experimental Procedures") with increasing concentrations of bovine brain cytosol either in the absence (bullet ) or in the presence of GTPgamma S (black-triangle) or AlF4- (black-square). After incubation, incorporation of [3H]UDP-GlcNac into VSV G protein was measured as a marker for transport.

To determine the topological orientation of heterotrimeric G proteins on Golgi membranes, we took the Galpha i3 subunit of G proteins as a representative of the Galpha class of proteins. The Galpha i3 subunit of heterotrimeric G proteins is localized to the cis side of Golgi stacks (9, 29). As shown in Fig. 3, more than 80% of membrane-bound Galpha i3 was digested by trypsin in the absence of detergent. In contrast, only the carboxyl-terminal fragment (~1 kDa) was removed from p23, a Golgi-localized type I membrane protein with a short cytoplasmic tail, showing that the large luminal domain of p23 was not accessible to trypsin and that the membranes are sealed under these conditions. In a control experiment it was shown that the luminal domain can in principle be digested by trypsin in the presence of detergent (Fig. 3, lane 4). Thus, with intact (sealed) membranes, Galpha i3 is accessible to trypsin and therefore must have a cytosolic orientation.


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Fig. 3.   G proteins are localized to the cytosolic face of Golgi membranes. Isolated Golgi membranes (lane 1, Input) were digested with trypsin (lane 2) or mock-digested with trypsin in the presence of soybean trypsin inhibitor (STI) (lane 3). As a control, Golgi membranes were digested with trypsin in the presence of 1% Triton X-100 (TX) (lane 4). Incubations and analysis were performed as described under "Experimental Procedures."

The inhibitory effect of AlF4- in the cell-free system was further investigated, and the formation of transport intermediates resistant to GTPgamma S and NEM (8) was compared with those formed in the presence of AlF4-. To this end, incubation of the cell-free system was started at t = 0 min, the transport inhibitors were added at t = 20 min, and the reaction was continued to completeness (60 min). Thus, if the inhibitor acts at an early step in the vesicle transport route (e.g. budding), then it will not act on the vesicles that have already formed (but not fused yet) during the 20-min incubation. These vesicles can then still fuse during the remaining time of incubation and result in a signal. On the other hand, if the inhibitor acts late (e.g. in a fusion process), most of the vesicles that have already formed after 20 min can still be prevented from fusing with the acceptor membrane. An incubation without inhibitors represents the maximal possible signal (Fig. 4, first lane). When samples are put on ice after 20 min, this represents the minimal signal i.e. a complete and instant block after 20 min of incubation (Fig. 4, second lane). Under these conditions, vesicles that have already formed are not able to fuse with the acceptor membrane. Any signal above the minimal signal represents the presence of a transport intermediate, resistant to the inhibitor. When NEM or GTPgamma S were added at t = 20 min, most of the vesicles that had formed during this time were still inhibited with NEM and to a significantly lower extent with GTPgamma S, indicating a late and an early inhibition step, respectively (Ref. 8 and Fig. 4, third and fourth lanes). A similar experiment with AlF4- (Fig. 4, fifth lane) resulted in a transport intermediate resistant to AlF4-, which is intermediate with respect to GTPgamma S and NEM, indicating that a G protein acts after binding of ARF to Golgi membranes (GTPgamma S effect) but before the NSF-dependent fusion step of COPI-coated vesicles with the acceptor membranes (NEM effect).


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Fig. 4.   AlF4- acts between the GTPgamma s- and NEM-sensitive transport steps. Transport assays were performed under standard conditions (see "Experimental Procedures"). As a control, transport of VSV G protein was measured after a 1-h incubation at 37 °C (1st lane). Parallel incubations were initiated at 37 °C, and after 20 min, the samples were put on ice (2nd lane) or inhibitors were added (as indicated in the 3rd-5th lanes), and the reaction was continued at 37 °C for 40 min (1 h total incubation). The error bars represent the mean average of 5 incubations.

These results raised the possibility that heterotrimeric G proteins are localized to COPI-coated vesicles and from there exert their inhibitory action. To determine the presence of G proteins on COPI-coated vesicles, we isolated these vesicles from a large scale incubation of Golgi membranes in the cell-free system in the presence of GTPgamma S. The total protein content of the vesicles cannot be used for quantitation because this also includes contamination of the vesicle preparation with relatively high amounts of actin and tubulin, resulting in an overestimation of the amount of vesicles. Therefore the vesicles were quantified based on their lipid (PC) composition. This lipid is present in relatively high amounts in membranes and is not expected to be sorted within the Golgi stack (30). Fig. 5 shows a comparison of equal amounts of donor Golgi and COPI-coated vesicles, based on their phosphatidylcholine content. p23, a Golgi-localized protein, is enriched in Golgi membranes and becomes more concentrated in COPI-coated vesicles, in agreement with previous observations (18). In addition, beta '-COP, a subunit of the coatomer complex, is enriched in the vesicle fraction, as expected (Fig. 5). Antibodies against various sub-classes of heterotrimeric G proteins were then used to identify the presence of these G proteins on the vesicles. Most of the antibodies do detect the presence of G proteins on the donor Golgi membranes, except for antibodies raised against the Galpha o subclass. In contrast, these antibodies could not detect the presence of G proteins in the purified COPI-vesicle fraction derived from the donor Golgi membranes, including antibodies against the Galpha s-, Galpha i-, and Galpha o- subclass. Antibodies against the Galpha q/11 and Galpha 12 subclass of G proteins detected small amounts of their antigens in COPI-coated vesicles but clearly in much smaller amounts as compared with the donor membranes. Likewise, an antibody against a common motif of various Gbeta isoforms failed to reveal any of the Golgi-localized isoforms in COPI-coated vesicles. Also under conditions where vesicles were generated in the presence of both GTPgamma S and AlF4- (to activate G proteins during the generation of COPI-coated vesicles) are G proteins strongly segregated from COPI-coated vesicles (Fig. 5, lane 4).


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Fig. 5.   Heterotrimeric G proteins are excluded from COPI-coated vesicles. CHO cells were homogenized, and Golgi membranes were isolated from these homogenates as described under "Experimental Procedures." COPI-coated vesicles were generated by incubation in the cell-free system of the Golgi membranes (see "Experimental Procedures") in the presence of GTPgamma S or GTPgamma S and AlF4-. Golgi membranes and COPI-coated vesicles were quantified by electrospray ionization tandem mass spectrometry (see "Experimental Procedures"). The samples were analyzed by SDS-PAGE (16) and subsequent Western blotting. Lane 1, homogenate (Hom) (10 µg of total protein); lane 2, CHO Golgi (10 µg of total protein, corresponding to 3.0 µg of PC); lane 3, COPI-coated vesicles (Ves) generated in the presence of GTPgamma S (containing 3.0 µg of PC); lane 4, COPI-coated vesicles generated in the presence of GTPgamma S and AlF4- (containing 2.5 µg of PC).

G Proteins Are Present in a Detergent-insoluble Complex-- What could be the basis for this exclusion of heterotrimeric G proteins from COPI-coated vesicles? Because G proteins do not have an obvious sorting, retention, or retrieval signal, we reasoned that other interaction(s) would be the most likely explanation for Golgi localization of heterotrimeric G proteins and their segregation from COPI-coated vesicles. We therefore analyzed Golgi membranes after mild detergent-treatment for the presence of complexes that contain G proteins. Several different detergents were tested for their inability to solubilize G proteins from Golgi membranes under conditions that lead to solubilization of Golgi membranes. Solubilization of Golgi membranes with Triton X-100 or CHAPS and subsequent centrifugation resulted in a detergent-insoluble fraction containing ~50 and ~20% of total Galpha i3, respectively (Fig. 6A, lanes 4 and 6). Other detergents such as octylglucoside, sodium deoxycholate (Fig. 6A, lanes 5 and 7) and cholate (Fig. 6B, lane 4) almost quantitatively solubilized the endogenous Galpha i3 and Gbeta gamma subunits. Conditions for detergent insolubility in Triton X-100 were further investigated by solubilization of Golgi membranes under various conditions. As shown in Fig. 6B, the presence of AlF4- did not alter the insolubility of Galpha i3, indicating that its presence in the detergent-insoluble fraction is independent of its state of activity. The presence of Galpha i3 in the detergent-insoluble fraction is, however, affected by the presence of divalent cations. In the presence of Mg2+, hardly any Galpha i3 could be recovered in the detergent-insoluble fraction (Fig. 6B, lane 8). These data indicate that the Galpha i3-containing detergent-insoluble fraction is different from another Golgi-localized detergent insoluble fraction, the so-called Golgi matrix (31), which is isolated from Golgi membranes by use of Triton X-100 and in the presence of Mg2+. It has been shown that high salt concentrations disrupt the Golgi matrix (32). In contrast, the addition of high salt did not disrupt the detergent-insoluble complex (Fig. 6B, lane 7). 40-50% of Galpha i3 remains associated with the detergent-insoluble pellet under these conditions, further indicating that this complex is distinct from the Golgi matrix.


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Fig. 6.   Heterotrimeric G proteins are present in detergent-insoluble Golgi fractions. CHO Golgi membranes (A and B) or a total membrane fraction from NRK cells (B) were solubilized in various detergent-containing buffers, and the detergent-insoluble fraction was isolated as described under "Experimental Procedures." The incubations were analyzed by SDS-PAGE and subsequent Western blotting using the indicated antibodies. A, lanes 1-3, CHO Golgi membranes without solubilization (lane 1, 1.25 µg or 25% of input; lane 2, 2.5 µg or 50% of input; lane 3, 5 µg or 100% of input); lanes 4-7, detergent-insoluble fraction from CHO Golgi membranes after solubilization in the following buffers: lane 4, 1% Triton X-100 in PEN buffer; lane 5, 60 mM octylglucoside in PEN buffer; lane 6, 20 mM CHAPS in PEN buffer; lane 7, 9 mM desoxycholate in 25 mM Hepes, 150 mM NaCl, pH 7.5. B, lanes 1-3, CHO Golgi membranes (upper panel, 5 µg of total protein) or NRK total membranes (lower panel, 20 µg of protein) without solubilization (lane 1, 25% of input; lane 2, 50% of input; lane 3, 100% of input); Lanes 4-9, detergent-insoluble fraction from CHO Golgi membranes or NRK total membrane fraction after solubilization in the following buffers: lane 4, 1% cholate in 10 mM Hepes, pH 8.0, 20 mM beta -mercaptoethanol, 1 mM EDTA (conditions used to solubilize and purify G proteins); lane 5, 1% Triton X-100 in PEN buffer; lane 6, same as lane 5 but in the presence of AlF4-; lane 7, same as lane 5 but in the presence of 500 mM NaCl; lane 8, 1% Triton X-100 in 50 mM MOPS, pH 7.0, 0.1 mM Mg2+, 1 mM dithiothreitol, and 10% sucrose (conditions used to isolate the Golgi matrix (31)); lane 9, same as lane 8 but in the presence of 500 mM NaCl to disrupt the Golgi matrix (32). CHO Golgi membranes and a total membrane fraction from NRK cells were prepared as described under "Experimental Procedures."

Our isolated Golgi membranes are contaminated with approximately 5-10% plasma membrane.2 We could not exclude the possibility that the insolubility of Galpha i3 is due to the presence of this protein in caveolae, which are present in the plasma membrane and which exhibit similar insolubility properties (33, 34). We therefore analyzed the characteristics of detergent insolubility of Galpha i3 present in NRK cells as well. In this cell-line, Galpha i3 is predominantly localized to Golgi membranes, and no plasma membrane localization could be observed (9). As shown in Fig. 6B, the characteristics of insolubility in Triton X-100 are very similar when comparing CHO Golgi membranes with a membrane fraction from NRK cells.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Reconstitution of intra-Golgi protein transport revealed the presence of both a GTPgamma S- and an AlF4--sensitive factor that inhibits the transport of the VSV G protein and results in an accumulation of COPI-coated vesicles (8). The GTPgamma S-sensitive factor was previously identified as a member of the ARF-family (2, 27), which in its purified form cannot be activated by AlF4- (7). Recently, it was shown that AlF4- can act on small molecular weight GTP-binding proteins such as Ras when it associates with its corresponding GTPase-activating protein (35, 36). By analogy, ARF complexed to its GTPase-activating protein might be a potential target for AlF4- in our in vitro assay system. In this case, GTPgamma S and AlF4- would act on the same protein (complex). The experiments described here, however, make it unlikely that AlF4- mimics the GTPgamma S effect for several reasons. (i) In contrast to ARF, the AlF4--sensitive factor resides on Golgi membranes (Fig. 2), (ii) whereas ARF is involved in the recruitment of coatomer to Golgi membranes (3, 4, 37) and uncoating of COPI-coated vesicles (5), the AlF4--sensitive factor might be involved in the actual fusion step (Fig. 2). With the transport assay, one can differentiate between vesicle-mediated transport and uncoupled fusion. The latter likely reflects the actual fusion step as soluble NSF attachment protein proteins and NSF are involved in this process (38). The uncoupled fusion reaction dominates in the transport assay under conditions of limited amounts of cytosol, which results in limited amounts of the coat proteins ARF and coatomer. As shown in Fig. 2, with reduced amounts of cytosol, GTPgamma S does not inhibit the transport assay, and this signal reflects uncoupled fusion. Thus, under transport (or rather fusion) conditions that are independent of ARF, AlF4- does inhibit the transport assay as effectively as under conditions of vesicle-mediated transport. These data show that the AlF4- effect is distinct from ARF. We also take these data as preliminary evidence for the involvement of an AlF4--sensitive factor in the fusion reaction. In agreement with this, we find that AlF4- also inhibits the vesicle-mediated reaction in the presence of saturating amounts of cytosol (Fig. 2), and (iii) determination of transport resistant-intermediates also shows a difference between GTPgamma S and AlF4-, with the AlF4- inhibition occurring after the GTPgamma S inhibition (Fig. 4). Surprisingly, the AlF4--resistant intermediate occurs before the NEM-resistant intermediate, suggesting that AlF4- inhibits before the action of NSF, which itself is part of the fusion reaction. NSF is part of a 20 S particle containing the cognate v- and t-SNARE proteins, and hydrolysis of its bound ATP results in disassembly of the SNARE complex (39). Therefore it is tempting to speculate that the AlF4--sensitive factor is involved in the assembly of the SNARE complex. This can be either during the actual recognition of a v-SNARE and its corresponding t-SNARE (docking reaction) or during the recruitment of v-SNAREs into transport vesicles. It cannot be excluded that the actual target of AlF4- is not a heterotrimeric G protein but rather a phosphate-binding protein like phosphatases (40). Phosphatases are generally inhibited by NaF itself in the millimolar range, without the addition of aluminum (41). Inhibition of the transport assay, however, is absolutely dependent on the presence of both F- and Al3+-ions (8).

Recently, heterotrimeric G proteins have been implicated in the regulation of the Golgi structure (12, 13). In contrast to our data indicating that G proteins act downstream of ARF, it was speculated that these G proteins act upstream in the process of COPI vesicle formation (13). Thus, it is possible that trimeric G proteins act at multiple stages in the secretory pathway. Alternatively, the G protein-mediated signaling pathways involved in the maintenance of the Golgi structure operate independently of G proteins involved in protein secretion.

Segregation of Golgi-localized G Proteins from COPI-coated Vesicles-- To determine the presence of G proteins in COPI-coated vesicles, we compared equal amounts of Golgi membranes with COPI-coated vesicles based on their PC content. We find that the segregation of trimeric G proteins from COPI-coated vesicles seems more efficient than has been described for Golgi-resident proteins (42).

The experimental system does not allow for discrimination between G proteins in their inactive (GDP) and active (GTP) conformation. GTPgamma S is needed to generate sufficient amounts of COPI-coated vesicles for a solid quantification of the G protein subunits. The portion of G proteins activated under our experimental conditions is unknown. Therefore, we used AlF4- to activate the complete pool of trimeric G proteins. This condition did not affect the segregation of trimeric G proteins from COPI-coated vesicles (Fig. 5), indicating that it is the activated form of trimeric G proteins that are segregated from COPI-coated vesicles. It cannot be excluded, however, that the inactive, GDP-bound trimeric G proteins enter the vesicles and are not segregated.

The efficiency of segregation might be overestimated for some G proteins. For example, Galpha s is predominantly localized to the trans-Golgi and trans-Golgi network in exocrine pancreas (29). Because COPI-coated vesicles are predominantly generated from the cis-, medial-, and trans-face of the Golgi stack (43) but not from the trans-Golgi network, Galpha s might simply not be present in the donor membranes at the site of vesicle formation. Galpha i3 and Galpha q/11 have been localized to the cis/medial Golgi cisternae (9, 29), and therefore these subunits must have been segregated during the generation of COPI-coated vesicles. The exact intracellular localization of the other G proteins tested for their segregation remains to be determined.

Are G Proteins Segregated from COPI-coated Vesicles by Specific Interactions with Other Proteins?-- We used Galpha i3 as a marker for cis-Golgi-localized trimeric G proteins and found that it is present in a detergent-insoluble complex. This indicates that Galpha i3 interacts with other proteins and that this interaction is not disrupted by some detergents. Galpha i3 is present in the detergent-insoluble pellet, irrespective of pretreatment of the Golgi membranes with GDP, AlF4- (Fig. 6), or with GTPgamma S (data not shown). This correlates well with the finding that trimeric G proteins are segregated from the vesicles in the absence and presence of AlF4-(Fig. 5).

How is Galpha i3 retained in the detergent-insoluble complex? A critical observation is that stimulation of G proteins with AlF4- does not change its participation in detergent-insoluble fractions (Fig. 6). This excludes the possibility that the G protein subunit beta gamma is responsible for the detergent insolubility of Galpha i3, because this subunit only interacts with Galpha in its inactive (GDP) state (44). Another possibility is that Golgi-localized G proteins are present in a detergent-insoluble complex due to their acylation with myristate and palmitate. Amino-terminal dual acylation of Galpha i3 with myristate and palmitate has been reported to be a determinant for caveolar localization (45, 46). Acylation is unlikely to be the sole determinant, since palmitoylation is a dynamic posttranslational event, and stimulation of Galpha subunits results in depalmitoylation (47, 48). Activation of Galpha with AlF4- also results in de-palmitoylation of the Galpha -subunit (48), yet its detergent insolubility is not affected (Fig. 6). Finally, these general mechanisms such as interaction with caveolae or Gbeta gamma or dual acylation with myristate and palmitate cannot explain the presence of Galpha subunits in the Golgi complex but rather would target these proteins to the plasma membrane. Given these considerations, it is likely that specific protein-protein interactions cause the presence of Golgi-localized G proteins in a detergent-insoluble complex. We are currently purifying this complex to homogeneity. Identification of proteins that interact with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might give an indication on the G protein-mediated signal transduction involved in protein secretion.

    ACKNOWLEDGEMENTS

We acknowledge Dr. J. E. Rothman for providing support during the early stages of this research, as it was initiated by J. B. H. as a postdoctoral fellow in his laboratory. We thank members of the Wieland lab for helpful discussions during the course of this work.

    FOOTNOTES

* This work was supported by a grant from the Deutsche Forschungsgemeinschaft and of the Human Frontier Science Program Organization.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.

§ Recipient of a fellowship from the Alexander von Humboldt Foundation. To whom correspondence should be addressed. Tel.: 49-6221-544688; Fax: 49-6221-544366; E-mail: Helms{at}urz.uni-heidelberg.de.

1 The abbreviations used are: G protein, heterotrimeric GTP-binding protein; CHO, Chinese hamster ovary; NRK, normal rat kidney; PC, phosphatidylcholine; VSV, vesicular stomatitis virus; NEM, N-ethylmaleimide; ARF, ADP-ribosylation factor; GTPgamma S, guanosine 5'-O-(thiotriphosphate); hCgB-GFP, green fluorescent protein-tagged human chromogranin B; PAGE, polyacrylamide gel electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; NSF, NEM-sensitive factor.

2 B. Brügger, unpublished data.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
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