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VCPIP1 binds p97. 38 / 38
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"The next question was, which region of p97 interacts with the two distinct binding sites in VCIP135?"
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"These results strongly suggest that VCIP135 can associate with p97 via two distinct interactions."
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"We next aimed to generate mutants of these two p97-binding regions in VCIP135 that do not bind to p97."
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"Full-length VCIP135 associates with p97 through their two distinct interactions."
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"As shown in xref B , the complex of VCIP135(F1024L/L1031P) and p97 was able to associate with another VCIP135 molecule (lane 3)."
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"These results indicate that p97 can associate with two VCIP135 molecules using the two binding interactions."
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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."
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"We also investigated the nucleotide dependency of the two interactions between VCIP135 and p97 using these full-length VCIP135 mutants."
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"Although VCIP135 also binds to p97 [13] , it remains unclear whether complex formation between VCIP135 and p97 is really necessary for p97-mediated membrane fusion."
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"We also showed that the binding of VCIP135 to p97 does not change its deubiquitinating activity ( xref )."
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"Additionally, the binding of VCIP135 to p97 was reported to cause no change in its deubiquitinating activity ( xref )."
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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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"The existence of these two binding interactions between p97 and VCIP135 has demonstrated several interesting points."
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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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"Although VCIP135 can bind to p97 in cytosol ( xref A), VCIP135 bound to syntaxin5 does not form a complex with p97 ( xref C, lane 4)."
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"We first tested whether phosphorylated VCIP135 binds to p97."
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"However, it remains unclear whether the interactions between VCIP135 and p97 are required for their biogenesis."
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"The phosphorylation of Ser-130 might disrupt the binding of p97 to the N-terminal binding site in VCIP135."
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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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"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."
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"Finally, we will discuss the functional significance of these two binding interactions in the VCIP135-p97 complex."
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"We established that VCIP135 is mitotically phosphorylated at Threonine-760 and Serine-767 and that the phosphorylated VCIP135 cannot bind to p97."
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"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. xref A shows that the addition of AMP-PNP, a nonhydrolyzable ATP analogue, increased the binding of VCIP135 to p97 (lane 4; xref A ), suggesting that ATP binding enhances the association between p97 and VCIP135."
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"We hence performed the following p97-VCIP135 binding experiments in the presence of AMP-PNP."
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"VCPIP1 interacts with p97, a highly abundant protein implicated in a vast range of cellular activities including the endoplasmic reticulum-associated degradation (ERAD) pathway and HIF1α stability xref ."
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"Surprisingly, both VCIP135 fragments bound to p97, and the binding efficiency of the N-terminal half was higher than that of the C-terminal half."
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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 ( xref D )."
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"We hence aimed to clarify the in vivo roles of the two binding interactions between VCIP135 and p97."
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"After incubation, the p97 bound to p47 was coimmunoprecipitated by anti-p47 antibodies in order to separate it from the p97 bound to VCIP135."
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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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"These data indicate that VCIP135 can form a stable complex with p97 in cytosol and that p47 is not part of this complex."
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"Phosphomimetic mutants at known VCIP135 phosphorylation sites prevent the association of VCIP135 with p97 [ xref ]."
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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 ( xref , xref )."
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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."
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"We also previously reported that both VCIP135 and p97 bind to ER membranes and function in ER network formation ( xref , xref )."
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