Two binding interactions in the p97/p47 complex
To determine the p97-binding sites on p47, we prepared several GST-tagged deletion mutants of p47 and checked their ability to bind to p97 ( A). p47(171–270) and p47(271–370) bind to p97, but p47(1–170) does not, suggesting the existence of two p97-binding sites on p47. This was confirmed by competition experiments between full-length p47 (p47full) and p47 fragments ( B). p47(171–270) and p47(271–370) inhibit the binding of p47full to p97, but p47(1–170) does not. Since p47full binds to the NH2-terminal domain of p97 (unpublished data), more detailed mapping of the p47-binding sites in p97 was performed ( C). The two p47 fragments showed different binding properties to p97. Although a single NH2-terminal domain of p97(1–74) was sufficient for p47(171–270) binding ( C, top), both p97 NH2-terminal domains, p97(1–74) and p97(134–198), were necessary for p47(271–370) binding ( C, bottom).
Figure 1. Two distinct binding interactions in the p97/p47 complex. (A) Each GST-tagged deletion mutant of p47 was incubated with p97 in a buffer containing 0.1% Triton X-100 on ice, then isolated on glutathione beads, and bound proteins were fractionated by SDS-PAGE. (more ...)
To characterize further the binding, EM of negatively stained complexes of p97/p47 fragments was performed. As shown in D, the averaged projection images of the resultant complexes were different to that of p97 alone. Since the sizes of the p47 fragments are too small (~10 kD) to allow direct observation by EM, the observed changes in the EM images largely represent conformational changes in p97. These observations therefore suggest that p97 undergoes significant conformational changes upon p47 fragment binding. In summary, our data show that p47 and p97 have two distinct binding interactions.
Identification of VCIP135 as a p97/p47 complex–interacting protein
We first tried to isolate a p97/p47-interacting protein using p47full beads but could only isolate p97 (unpublished data). This suggested that an interacting protein, even if it existed, might form an unstable ternary complex with p97 and p47. The existence of two distinct p97/p47 binding interactions could result in transient or unstable complexes that use only one p97/p47 binding interaction ( A). Based on the idea that such unstable p97/p47 complexes could be a target for an interacting protein X, we next tried to isolate such a protein using small p47 fragments containing only one of the p97-binding sites as bait. The p47 fragments were immobilized on beads, mixed with rat liver cytosol, and bound proteins were analyzed. The results are shown in B. As expected, both p47 fragments bound to p97. Interestingly, another protein of 135kD (VCIP135) was isolated only using p47(171–270) beads ( B, asterisk). After Zn-staining, this protein band was excised from the gel and subjected to protein microsequencing by electrospray mass spectrometry. We obtained two partial peptide sequences that allowed us to clone a novel cDNA sequence corresponding to VCIP135. Its nucleotide sequence are available from the DNA data bank of Japan database under the accession no. AB045378. Kazusa DNA Research Group also reported the cDNA sequence of its human homologue (Nagase et al., 2001
). Western blotting using anti-VCIP135 antibodies showed that it was widely expressed all over the rat tissues (unpublished data).
Figure 2. Identification of VCIP135. (A) A working model of how a p97/p47-interacting protein (protein X) forms an unstable ternary complex with p97 and p47. (B) Purification of VCIP135 from rat liver cytosol. Each small p47 fragment with a p97-binding site was (more ...)
VCIP135 dissociates the p97/p47 complex through p97 ATP hydrolysis
To characterize the interactions between VCIP135 and the p97/p47 complex, detailed immunoprecipitation experiments were performed. As shown in A, anti-VCIP135 antibodies coimmunoprecipitated a protein of 97 kD from the cytosol (left), which was shown to be p97 by Western blotting (right middle). However, p47 could not be detected in these precipitates ( A, right bottom). These data indicate that VCIP135 can form a stable complex with p97 in cytosol and that p47 is not part of this complex. As presented in B, the fractionation of cytosol by sucrose-gradient sedimentation showed that VCIP135 distributed in lighter fractions (lanes 3–6) and in the p97-containing fractions (lanes 7–10), suggesting the existence of two populations of cytosolic VCIP135: one in complex with p97 and the other free of p97.
Figure 3. VCIP135 binds to and dissociates the p97/p47 complex. (A) Rat liver cytosol (3.5 mg total protein) was incubated with preimmune serum or anti-VCIP135 antibodies. The immunoprecipitates were fractionated by SDS-PAGE followed by staining with Coomassie (more ...)
We then mapped the interaction between VCIP135 and p97, the results of which are shown in C (see also Fig. S1 available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1
). Various truncated His tag VCIP135 fragments were tested for their ability to bind p97. The COOH-terminal region of VCIP135 (744–1221) proved to be important for p97 binding ( C, lane 3). Interestingly, fold recognition analysis of VCIP135 identified a region between residues 744 and 860 to have a similar fold to SUMO-1 (Bayer et al., 1998
), suggesting a ubiquitin-like structure for this part of VCIP135. Recently, a nuclear magnetic resonance spectroscopy study has shown that the p47 COOH-terminal domain (residues 270–370) is also a ubiquitin-like domain and that this region specifically binds to the NH2
-terminal domain of p97 (Yuan et al., 2001
). It is therefore plausible that both VCIP135 and p47 use homologous ubiquitin-like domains ( D) to bind to the same region of p97, suggesting that p47(271–370) competes with VCIP135 for p97 binding. This provides a reasonable explanation for our biochemical data that p47(271–370) binds to p97 but not to the p97/VCIP135 complex ( B).
We originally isolated VCIP135 as a candidate p97/p47-interacting protein and thus needed to establish whether it can form a complex with p97/p47. As discussed in the previous section, a ternary complex of p97/p47/VCIP135 might be unstable, and hence, we used a cross-linker reagent to capture it. Coimmunoprecipitation experiments were performed in the presence of the cross-linker, sulfosuccinimidyl 2-[m-azio-o-nitrobenzamido]ethyl-1,3′-dithiopropionate (SAND) ( E). VCIP135 was coimmunoprecipitated by anti-p47 antibodies only in the presence of SAND ( E, lanes 3 and 4) but not in its absence (lane 1). The preimmune serum did not coimmunoprecipitate any VCIP135 even in the presence of SAND ( E, lane 5). ufd1p, a negative control, was not precipitated in any lanes (bottom). Hence, VCIP135 could bind to the p97/p47 complex in cytosol, possibly forming an unstable complex of p47, p97, and VCIP135 that was captured after chemical cross-linking. This led to the question of what effect VCIP135 would have on the p97/p47 complex after forming the unstable ternary complex.
To answer this question, VCIP135 was added to the purified p97/p47 complex in the presence of nucleotides ( F). After incubation, the p97 bound to p47 was coimmunoprecipitated by anti-p47 antibodies in order to separate it from the p97 bound to VCIP135. In the presence of ATP, VCIP135 dissociated the p97/p47 complex in a dose-dependent manner ( F, left three lanes). In the presence of AMP–PNP, a nonhydrolyzable ATP analogue, or ADP, the dissociation of the complex was not observed ( F, the middle and right three lanes, respectively), indicating that dissociation of p97/p47 complex by VCIP135 required ATP hydrolysis. Together these data show that VCIP135 interacts with the p97/p47 complex and dissociates it as a consequence of ATP hydrolysis by p97.
VCIP135 dissociates the p97/p47/syntaxin5 complex
Although VCIP135 was originally purified from cytosol, it is also localized to Golgi and ER. As presented in , double immunofluorescence staining of VCIP135 and either β1,4-galactosyltransferase (GalT), a Golgi marker, or protein disulfide isomerase (PDI), an ER marker, showed localization to Golgi and ER.
Figure 4. The localization of VCIP135. Double immunofluorescence staining of VCIP135 and either GalT, a Golgi marker, or PDI, an ER marker, shows that VCIP135 localizes to Golgi and ER. NRK cells were fixed by methanol at −20°C for 4 min, stained (more ...)
Syntaxin5 is a SNARE in both the Golgi and ER (Hui et al., 1997
) and has been shown to be involved in the p97/p47 fusion pathway (Rabouille et al., 1998
). Hence, we investigated the interaction between VCIP135 and syntaxin5 by binding experiments using GST-tagged syntaxin5 without the transmembrane domain (GST–syn5ΔTM) as shown in A. Since syntaxin1 is a SNARE in plasma membranes, we used GST–syn1ΔTM as a negative control. We found that GST–syn5ΔTM bound VCIP135 very efficiently ( A, lane 1) compared with GST–syn1ΔTM (lane 2). The specificity of binding of VCIP135 to syntaxin5 was further confirmed by the coimmunoprecipitation experiments using Golgi membranes as shown in B. 1 M KCl-washed Golgi membranes were incubated with His-tagged VCIP135. After removing unbound VCIP135, the membranes were solubilized and VCIP135, and its binding proteins were immunoprecipitated by antibodies to the His tag on VCIP135. Syntaxin5 was coimmunoprecipitated ( B), providing evidence that VCIP135 binds to syntaxin5 in Golgi membranes.
Figure 5. VCIP135 binds to syntaxin5 and dissociates the p97/p47/syntaxin5 complex via p97 catalyzed ATP hydrolysis. (A) His-tagged VCIP135 (0.4 μg) was incubated with either GST, GST–syn5ΔTM, or GST–syn1ΔTM (0.5 μg (more ...)
We next incubated VCIP135 and GST–syn5ΔTM together with p97 and p47 in the absence of nucleotides and then isolated the proteins bound to GST–syn5ΔTM ( C). VCIP135 bound to GST–syn5ΔTM either with p47 or p97/p47 ( C, lanes 5 and 6, respectively). This simple binding experiment showed that VCIP135 can form a quaternary complex with syntaxin5, p97, and p47 (Fig. S2 available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1
). VCIP135 did not bind to syntaxin5 with p97 ( C, lane 4), a finding which was investigated further ( F).
A further question is what are the consequences of the ATP hydrolysis by p97 on the quaternary complex of VCIP135/p97/p47/syntaxin5? To address this, we first performed binding experiments: p97/p47 was incubated with either GST–syn5ΔTM or VCIP135 + GST–syn5ΔTM in the presence of nucleotides, and the proteins bound to GST–syn5ΔTM were analyzed ( D; see also Fig. S3 available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1
). In the presence of ATP, the addition of VCIP135 prevented p97 and p47 from binding to syntaxin5 and VCIP135 itself only bound poorly to syntaxin5 ( D, left three lanes). However, in the presence of AMP–PNP or ADP, p97 and p47 bound to syntaxin5 even when VCIP135 was added, and VCIP135 also bound to syntaxin5 ( D, right six lanes). These data suggest that both VCIP135 and the p97/p47 complex bind to syntaxin5, resulting in the formation of a transient quaternary complex that could be dissociated through p97 ATP hydrolysis. To clarify this further, we performed dissociation experiments as described in E (see also Fig. S4 available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1
). We initially prepared complexes of p97/p47/GST–syn5ΔTM or VCIP135/p97/p47/GST–syn5ΔTM in the presence of AMP–PNP. Both complexes were maintained under these conditions. Then, the beads containing both complexes were incubated in the presence of AMP–PNP or ATP. The p97/p47/GST–syn5ΔTM complex was maintained intact even in the presence of ATP ( E, lanes 1 and 2). However, the VCIP135/p97/p47/GST–syn5ΔTM complex was dissociated only in the presence of ATP ( E, lane 4), and the VCIP135/p97 complex was observed in the supernatant (unpublished data). This result strongly suggests that VCIP135 dissociates the p97/p47/syntaxin5 complex via ATP hydrolysis by p97.
Although VCIP135 can bind to p97 in cytosol ( A), VCIP135 bound to syntaxin5 does not form a complex with p97 ( C, lane 4). This suggests that the formation of a VCIP135/p97 complex on syntaxin5 dissociates VCIP135 from syntaxin5. To investigate this, we performed stripping experiments as shown in F. p97 was added to a complex of VCIP135/GST–syn5ΔTM, and the remaining amounts of VCIP135 on GST–syn5ΔTM were determined. The addition of p97 decreased the amount of VCIP135 bound to syntaxin5 ( F). The dissociated VCIP135 was observed as a complex with p97 in the supernatant (unpublished data).
We also investigated whether VCIP135 has any effect on the binding of α-SNAP to syntaxin5. As shown in G, VCIP135 partially prevented α-SNAP binding to GST–syn5ΔTM, suggesting that VCIP135 directs syntaxin5 to p97/p47 but not to α-SNAP/NSF.
VCIP135 is essential for p97/p47-mediated membrane fusion
We have shown that VCIP135 binds to syntaxin5 and disassembles the p97/p47/syntaxin5 complex through p97 ATP hydrolysis. Next, we wanted to ascertain whether VCIP135 contributes to p97/p47-mediated membrane fusion using an in vitro Golgi reassembly assay. As shown in A, cisternal regrowth was observed by the incubation of mitotic Golgi fragments with the p97/p47 complex (the control). Anti-VCIP135 antibodies inhibited the p97/p47-mediated cisternal regrowth by ~75% compared with the control. This inhibition was rescued by quenching the antibodies with a VCIP135 fragment. The antibodies had no effect on NSF-mediated cisternal regrowth ( B). Together, these results strongly suggest that VCIP135 is specifically involved in the p97/p47 membrane fusion pathway.
Figure 6. VCIP135 is essential for p97/p47-mediated membrane fusion. (A) Mitotic Golgi membranes were incubated with the indicated components at 37°C for 60 min. The membranes were fixed, processed, and the percentage of membrane in cisterna was determined. (more ...)
The above assays were performed with Golgi mitotic fragments that were contaminated by membrane-bound VCIP135. Therefore, we performed assays using VCIP135-free membranes obtained by 1 M KCl washes ( C, top). When the VCIP135-free membranes were incubated with p97/p47, VCIP135, or p97/VCIP135, no cisternal regrowth was observed. Only when p97/p47 and VCIP135 were added together was cisternal regrowth observed ( C). Thus, VCIP135 is an essential factor for p97/p47-mediated membrane fusion. D shows the dose dependency of VCIP135 on cisternal regrowth. Interestingly, it is biphasic: a low level of VCIP135 (1/8–1/2 [mol/mol p97]) had a positive effect on the cisternal regrowth, whereas a higher level of VCIP135 had a negative effect.
VCIP135 is required for Golgi and ER assembly in vivo
Although many groups have reported the in vitro function of p97 in the biogenesis of organelles, its in vivo function still remains unclear. Since p97 is known to be involved in several biological processes (Patel and Latterich, 1998
; Meyer et al., 2000
; Dai and Li, 2001
; Ye et al., 2001
; Jarosch et al., 2002
), a specific cofactor other than p97 would be a better target to mediate its fusion-specific function. We have now identified VCIP135 as an essential cofactor for the fusion activity of p97 in vitro. To study the function of VCIP135 in vivo, we microinjected anti-VCIP135 antibodies into cells and investigated its effects on the reassembly of organelles at the end of mitosis. Cells at prophase (or early prometaphase) were injected with anti-VCIP135 antibodies and fixed after they entered interphase. An anti-VCIP135 antibody quenched by its antigen and rabbit random IgG were injected as negative controls, and an anti-p47 antibody was used as a positive control. The injections of these antibodies had no effect on cell cycle progression.
As shown in A, the Golgi was stained by mAbs to GalT, a Golgi marker (Rabouille et al., 1995a
). Hoechst DNA staining showed that injected cells had exited mitosis (bottom). After the injection of either anti-VCIP135 or anti-p47 antibodies, Golgi still localized to a perinuclear region in daughter cells (top panel 2 and 4, respectively). The ultrastructure of the Golgi in injected cells was investigated by EM (, B–D). In anti-VCIP135 antibody-injected cells, highly organized Golgi complexes were lost: stacked cisternae were rarely observed (anti-VCIP135 antibody injection, 30.3% of cisternal membranes in stacks; random IgG injection, 80.4%), and the number of tubular/fenestrated structures was greatly increased ( B, top left). As shown in C, immuno-EM images using antibodies to Golgi marker proteins, GalT or GM130 (Nakamura et al., 1995
; Rabouille et al., 1995a
), showed that these fragmented membranes were derived from Golgi. The quantitative results are shown in D. The injection of anti-VCIP135 antibodies significantly decreased the percentage of cisternae per Golgi area by a half and significantly increased the percentage of tubules twofold compared with injections of quenched antibodies and random IgG. The amount of vesicles was not changed. Similar morphological changes were observed after the injection of anti-p47 antibodies ( B, left bottom, and D). Since the injection of the anti-VCIP135 antibodies at the same concentration into interphase cells caused no obvious changes in the organization of the Golgi apparatus (by IF and EM observation [unpublished data]), these morphological changes were most likely caused by the inhibition of reassembly at the end of mitosis. These data show that VCIP135 functions in the reassembly of highly organized Golgi structures in living cells as does p47, indicating that p97-mediated fusion is required for Golgi reassembly in living cells.
Figure 7. VCIP135 is required for Golgi assembly in vivo. (A) NRK cells at prophase (or early prometaphase) were injected with antibodies and fixed after they exited mitosis. Golgi (green, top), injected antibodies (red, top), and chromatin (bottom) were stained (more ...)
p97/p47 has also been implicated in ER membrane fusion in an in vitro system (Hetzer et al., 2001
). VCIP135 localizes to ER membranes and Golgi (, bottom). Hence, we investigated the effect of antibodies to VCIP135 on ER structures. As shown in A, the injection of anti-VCIP135 antibodies caused formation of a large meshed ER network in daughter cells (panel 2). Injection of anti-p47 antibodies had a similar effect ( A, panel 4). This change in ER structure was not observed after injecting quenching antibodies ( A, panel 3) or random IgG (panel 1). To quantify the degree of ER network formation, the number of three-way junctions was counted using confocal microscopy ( B). The number of three-way junctions was significantly decreased by ~70% compared with control by the injection of antibodies to VCIP135 and p47. These data indicate that the p97/p47/VCIP135 pathway also participates in ER network formation in living cells.
Figure 8. VCIP135 is required for ER network formation in vivo. (A) Antibodies were microinjected into CHO cells expressing GFP-tagged HSP47. Since ER structures were easily damaged and lost after fixation, they were observed in living cells without fixation by (more ...)
The structures of other organelles, including nuclear envelope, mitochondria, and lysosome, were also investigated by EM in the antibodies injected cells. After microinjection of either anti-VCIP135 or anti-p47 antibodies, no obvious morphological changes in these organelles were observed (unpublished data) in contrast with the changes seen in the structures of the Golgi and ER. Detailed images of the cells injected with anti-VCIP135 antibodies are shown in Figs. S5 and S6, available at http://www.jcb.org/cgi/content/full/jcb.200208112/DC1