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VCPIP1 binds VCP. 49 / 61
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"Overall, our study not only reveals the unexpected interaction and organization of human P97/VCP‐VCPIP1 complex, but also provides crucial insights into the mechanism of VCPIP1‐P97/VCP mediated Golgi biogenesis.2 Results."
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"To validate the direct interaction between VCPIP1 and P97/VCP in vitro, we first purified the full‐length proteins and analyzed their interactions by size exclusion chromatography."
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"The obtained result showed that the VCPIP1 could directly bind to P97/VCP by migrating together with P97/VCP on the size exclusion column (Figure 1B)."
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"The results reveal that, in the absence of substrates, the binding of VCPIP1 to P97/VCP led to a marginal increase in its activity (Figure 1E).2.2 Cryo-EM Structure of VCPIP1-P97/VCP Complex."
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"Albeit with the relatively lower and heterogeneous resolution of VCPIP1, the regions of VCPIP1 that directly contact P97/VCP could be readily and confidently modeled.Expectedly, the P97/VCP in the P97/VCP‐VCPIP1 complex was folded into the canonical homo‐hexamer as previously studied (Figure 2A,C)."
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"Unexpectedly, three VCPIP1 molecules directly bind to the C‐terminal D2 ATPase domain of the P97/VCP, resulting in the formation of P97/VCP‐VCPIP1 complex with a stoichiometry of 2:1 (Figure 2B)."
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"Since only one 3D reconstruction was obtained and could be refined to high resolution from our cryo‐EM studies, we favored it as the stable and dominant conformation adopted by the full‐length P97/VCP‐VCPIP1 complex (Figure S1C, Supporting Information)."
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"However, the ATPase assay revealed that VCPIP1 binding to P97/VCP only slightly increases its ATPase activity (Figure 1E).2."
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"Golgi WAC interacts with the deubiquitinase VCIP135, which binds VCP and p97."
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"The resulting structural model indicates that VCPIP1 binds to P97/VCP through three major interfaces, with both the N‐terminal and C‐terminal regions of P97/VCP required for VCPIP1 binding (Figure 4A)."
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"Previous studies have proved that VCPIP1 interaction with P97/VCP synergistically facilitates the post‐mitotic Golgi vesicle fusion by regulating the priming of syntaxin‐5 containing SNARE complex through the P97/VCP adaptor protein P47 during the cell cycle."
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"These biochemical assays further demonstrated that VCPIP1 contacts the D2 domain of P97/VCP through its UFD1 domain, inducing a conformational change in P97/VCP to favor its binding to the SNARE substrate, thereby regulating the disassembly of the SNARE complex.To further investigate the functional relevance of the P97/VCP‐VCPIP1 complex in post‐mitotic Golgi reassembly, we generated a VCPIP1‐knockout HeLa cell line using CRISPR‐Cas9 technology."
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"VCIP135 binds to the p97/p47/syntaxin5 complex and promotes its dissociation via p97 catalysed ATP hydrolysis ( Uchiyama et al., 2002 )."
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"In this study, our structural and functional studies of the P97/VCP‐VCPIP1 complex provide key insights into the organization and membrane fusion mechanism of the underappreciated P97/VCP‐VCPIP1 complex.VCPIP1 was initially identified as an essential factor for P97/VCP/p47‐mediated Golgi and ER assembly (15)."
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"We concluded that this UFD1‐mediated VCPIP1 interaction with P97/VCP is critical for their function in Golgi reassembly."
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"We determine a ∼3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer."
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"Whether the interaction between P97/VCP and VCPIP1 is directly regulated by CDK1 or other mitotic kinases needs further exploration."
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"34 Considering the key role in contact with VCP by VCPIP1 Y623 and F638, which are the neighboring residues of K632 and K646, UFD1 ubiquitination might have a direct effect on their interaction and thus regulate Golgi membrane dynamics.In conclusion, our findings uncover the high‐resolution structure of the P97/VCP‐VCPIP1 complex and reveal the unique and unexpected binding modes between the ATP‐driven substrate unfoldase P97/VCP and the deubiquitinase VCPIP1."
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"We determine ∼3.3 Å cryogenic electron microscopy structures of VCP-VCPIP1 complexes in the absence of added nucleotide or the presence of an ATP analog."
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"Additionally, the binding of VCIP135 to p97 was reported to cause no change in its deubiquitinating activity (9)."
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"To date, there are no lines of evidence indicating that VCIP135 requires its binding to p97 for Golgi and ER biogenesis.In this study, we investigated the binding interactions between VCIP135 and p97 and found that VCIP135 has two distinct p97-binding sites; that is, its N- and C-terminal p97-binding sites interact with the C- and N-terminal regions of p97, respectively."
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"As p97 is an ATPase, we first analyzed the effects of nucleotides on the binding between p97 and VCIP135."
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"Investigators show that VCPIP1 interacts with VCP and other genes associated with the reassembly of the Golgi apparatus and the endoplasmic reticulum during mitosis [ xref , xref ]."
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"Figure 1A shows that the addition of AMP-PNP, a nonhydrolyzable ATP analogue, increased the binding of VCIP135 to p97 (lane 4; Fig. S1A), suggesting that ATP binding enhances the association between p97 and VCIP135."
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"VCIP135(1-390) bound to p97, whereas VCIP135(361-743) did not."
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"Regarding the p97-binding sites in its C-terminal half, only VCIP135(903-1053) bound to p97, but the other fragments did not (Fig. 1D)."
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"These results strongly suggest that VCIP135 can associate with p97 via two distinct interactions."
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"VCIP135(1-743)wt bound to p97 (top panel, lane 2), whereas VCIP135(1-743)(L133S) showed almost no binding to p97 (top panel, lane 3)."
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"The VCIP135 mutants (L133S) and (F1024L/L1031P), in which only one of the two p97-binding sites was intact, still bound to p97, although their binding affinities were decreased compared with that of VCIP135wt (Fig. 5A, lanes 2–4; Fig. S1D)."
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"In other words, these results strongly support the fact that each of the two distinct binding interactions between VCIP135 and p97 is sufficient for VCIP135-p97 complex formation.We also investigated the nucleotide dependency of the two interactions between VCIP135 and p97 using these full-length VCIP135 mutants."
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"We determine a ∼3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer."
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"These results indicate that the binding of ATP to p97 enhances both of these interactions between VCIP135 and p97; that is, these two interactions in the complex may be synchronously controlled by the nucleotide state of p97."
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"The two interactions between VCIP135 and p97 are required for Golgi and ER biogenesis in vivo."
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"We previously reported that both VCIP135 and p97, which form a complex in the cytosol, bind to Golgi and ER membranes, and function in Golgi and ER biogenesis (6, 8)."
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"However, it remains unclear whether the interactions between VCIP135 and p97 are required for their biogenesis."
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"These in vivo results strongly suggest that both of the two interactions between VCIP135 and p97 are required for Golgi biogenesis.We also previously reported that both VCIP135 and p97 bind to ER membranes and function in ER network formation (8, 9)."
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"P97 binds a variety of proteins with various functions, for example VCIP135 (valosin-containing protein (p97)/p47 complex-interacting protein p135) [3] and p47."
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"We therefore investigated the roles of the interactions between VCIP135 and p97 in ER network formation in living cells."
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"Third, these two binding interactions in the complex are synchronously controlled by the nucleotide state of p97 (Fig. 5, C and D), which leads to the nucleotide-dependent association between full-length VCIP135 and p97 (Fig. 1A)."
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"Finally, each of the two binding interactions between VCIP135 and p97 is sufficient for their complex formation."
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"The phosphorylation of Ser-130 might disrupt the binding of p97 to the N-terminal binding site in VCIP135.In the present study, we clarified that VCIP135 and p97 form a complex through two distinct interactions and that either one of the interactions is sufficient for this complex formation."
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"This indicates that the association between p97 and VCIP135 is necessary for Golgi and ER biogenesis."
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"Interestingly, these morphological changes were not rescued by the expression of VCIP135 mutants with only one of the two p97 binding sites, indicating that these two distinct interactions between VCIP135 and p97 are essential for Golgi and ER biogenesis."
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"We also showed that the binding of VCIP135 to p97 does not change its deubiquitinating activity (9)."
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"Taking these facts altogether, it is plausible that these binding interactions of VCIP135 with p97 may be required for the ubiquitin-independent functions of VCIP135, but not for its ubiquitin-dependent function.Finally, we will discuss the functional significance of these two binding interactions in the VCIP135-p97 complex."
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"The tethering complex is generally thought to be dissociated and then reused after membrane fusion, and hence VCIP135 might be a candidate recycling factor of the tethering complex.Another important point is that VCIP135 can associate with p97 by binding to either its N- or C-terminal region."
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"Conversely, VCPIP1 bound P97/VCP hexamer favors the binding of P47, and thus the intact SNARE complex, promoting Golgi membrane fusion."
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"28 However, the molecular mechanism of VCPIP1 interacting with P97/VCP and the precise roles of the VCPIP1‐P97/VCP complex in Golgi membrane fusion in late mitosis is still elusive.In this study, we first analyzed the interactions between P97/VCP and VCPIP1 fragments and found out that VCPIP1 can associate with P97/VCP in vitro through multiple binding sites, including the N‐terminal OTU domain, the middle UFD1 domain, and the C‐terminal UBL‐UFD2 domain."
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"Moreover, we successfully determined the Cryo‐EM structure of the full‐length P97/VCP‐VCPIP1 complex, which reveals that three individual VCPIP1 molecules directly bind to the C‐terminal D2 domains of the P97/VCP hexamer through their UFD1 domains."
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