VCIP135 deubiquitinase and its binding protein, WAC, in p97ATPase-mediated membrane fusion

Two distinct p97 membrane fusion pathways are required for Golgi biogenesis: the p97/p47 and p97/p37 pathways. VCIP135 is necessary for both pathways, while its deubiquitinating activity is required only for the p97/p47 pathway. We have now identified a novel VCIP135-binding protein, WAC. WAC localizes to the Golgi as well as the nucleus. In Golgi membranes, WAC is involved in a complex containing VCIP135 and p97. WAC directly binds to VCIP135 and increases its deubiquitinating activity. siRNA experiments revealed that WAC is required for Golgi biogenesis. In an in vitro Golgi reformation assay, WAC was necessary only for p97/p47-mediated Golgi reassembly, but not for p97/p37-mediated reassembly. WAC is hence thought to function in p97/p47-mediated Golgi membrane fusion by activating the deubiquitinating function of VCIP135. We also showed that the two p97 pathways function in ER membrane fusion as well. An in vitro ER reformation assay revealed that both pathways required VCIP135 but not its deubiquitinating activity for their ER membrane fusion. This was consistent with the finding that WAC is unnecessary for p97-mediated ER membrane fusion.     

Cell cycle regulation of VCIP135 deubiquitinase activity and function in p97/p47-mediated Golgi reassembly

In mammalian cells, the inheritance of the Golgi apparatus into the daughter cells during each cycle of cell division is mediated by a disassembly and reassembly process, and this process is precisely controlled by phosphorylation and ubiquitination. VCIP135 (valosin-containing protein p97/p47 complex–interacting protein, p135), a deubiquitinating enzyme required for p97/p47-mediated postmitotic Golgi membrane fusion, is phosphorylated at multiple sites during mitosis. However, whether phosphorylation directly regulates VCIP135 deubiquitinase activity and Golgi membrane fusion in the cell cycle remains unknown. We show that, in early mitosis, phosphorylation of VCIP135 by Cdk1 at a single residue, S130, is sufficient to inactivate the enzyme and inhibit p97/p47-mediated Golgi membrane fusion. At the end of mitosis, VCIP135 S130 is dephosphorylated, which is accompanied by the recovery of its deubiquitinase activity and Golgi reassembly. Our results demonstrate that phosphorylation and ubiquitination are coordinated via VCIP135 to control Golgi membrane dynamics in the cell cycle.     

p97/p47-Mediated biogenesis of Golgi and ER

In mammalian cells, the Golgi apparatus and endoplasmic reticulum have typical structures during interphase: stacked cisternae located adjacent to the nucleus and a network of interconnected tubules throughout the cytoplasm, respectively. At mitosis their architectures disappear and are reassembled in daughter cells. p97, an AAA-ATPase, mediates membrane fusion and is required for reassembly of these organelles. In the p97-mediated membrane fusion, p47 was identified as an essential cofactor, through which p97 binds to a SNARE, syntaxin5. A second essential cofactor, VCIP135, was identified as a p97/p47/syntaxin5-interacting protein. Several lines of recent evidence suggest that ubiquitination may be implicated in the p97/p47 pathway; p47 binds to monoubiquitinated proteins and VCIP135 shows a deubiquitinating activity in vitro. For the cell-cycle regulation of the p97/p47 pathway, it has been reported that the localization and phosphorylation-dephosphorylation of p47 are crucial. In this review, we describe the components involved in the p97-mediated membrane fusion and discuss the regulation of the fusion pathway.     

The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.     

p97 and p47 function in membrane tethering in cooperation with FTCD during mitotic Golgi reassembly

p97ATPase‐mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N‐ethylmaleimide‐sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47‐mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47‐binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47‐mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD‐p97/p47‐FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD‐p97/p47‐FTCD complex in p97/p47‐mediated Golgi membrane fusion.     

Conformational changes in the AAA ATPase p97-p47 adaptor complex

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.     

VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments

The AAA-ATPase p97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1-Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin-proteasome system is also involved in membrane fusion events. Here, we show that p97-p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of p97/Cdc48.     

A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.     

p97 adaptor choice regulates organelle biogenesis

The AAA protein p97 requires adaptor-like cofactors for its numerous cellular functions. In this issue of Developmental Cell, Uchiyama et al. (2006) identify p37 as a p97 adaptor that is required constitutively for ER and Golgi membrane fusion, analogous to the mitotic membrane fusion role of the adaptor p47. Their study suggests that related p97 adaptors involved in similar cellular pathways can be subject to differential regulation.     

Phospholipid species act as modulators in p97/p47-mediated fusion of Golgi membranes

The ATPase p97 in complex with p47 participates in Golgi cisternae rebuilding after mitosis. In a Golgi-liposome assay, the complex triggered a phosphatidylethanolamine (PE)-promoted fusion. Here we show for the first time that fusion between mitotic Golgi membranes induced by adding cytosol or purified p97/p47 is modulated by PE present in Golgi membranes. Using model membranes, we demonstrate a PE-dependent recruitment of p97/p47 to membranes, causing dramatic conformational rearrangements and favoring protein-lipid interactions. Previously buried hydrophobic sites become exposed in a controlled manner, which leads to the penetration of (a) domain(s) of the complex into lipid bilayers, facilitated by a PE-dependent increase in headgroup spacing. In contrast, when facing phosphatidylcholine (PC) the complex clusters extensively. This implies that in the presence of PC protein-protein interactions rather than fusion-promoting protein-lipid interactions occur. Importantly, PE-mediated changes in secondary and tertiary structures are exclusively observed when p97 is complexed with p47, which is a prerequisite for membrane fusion. We therefore propose that at physiological conditions PE-induced conformational changes in p97/p47 are relevant in triggering this activity.     

VCIP135, a novel essential factor for p97/p47-mediated membrane fusion,is required for Golgi and ER assembly in vivo

NSF and p97 are ATPases required for the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of organelles. NSF uses ATP hydrolysis to dissociate NSF/SNAPs/SNAREs complexes, separating the v- and t-SNAREs, which are then primed for subsequent rounds of fusion. In contrast, p97 does not dissociate the p97/p47/SNARE complex even in the presence of ATP. Now we have identified a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), and show that it binds to the p97/p47/syntaxin5 complex and dissociates it via p97 catalyzed ATP hydrolysis. In living cells, VCIP135 and p47 are shown to function in Golgi and ER assembly.     

Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes,p47 and Ufd1-Npl4

The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.     

ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

Cell cycle maintenance and biogenesis of the Golgi complex

How organelle identity is established and maintained, and how organelles divide and partition between daughter cells, are central questions of organelle biology. For the membrane-bound organelles of the secretory and endocytic pathways [including the endoplasmic reticulum (ER), Golgi complex, lysosomes, and endosomes], answering these questions has proved difficult because these organelles undergo continuous exchange of material. As a result, many "resident" proteins are not localized to a single site, organelle boundaries overlap, and when interorganellar membrane flow is interrupted, organelle structure is altered. The existence and identity of these organelles, therefore, appears to be a product of the dynamic processes of membrane trafficking and sorting. This is particularly true for the Golgi complex, which resides and functions at the crossroads of the secretory pathway. The Golgi receives newly synthesized proteins from the ER, covalently modifies them, and then distributes them to various final destinations within the cell. In addition, the Golgi recycles selected components back to the ER. These activities result from the Golgi's distinctive membranes, which are organized as polarized stacks (cis to trans) of flattened cisternae surrounded by tubules and vesicles. Golgi membranes are highly dynamic despite their characteristic organization and morphology, undergoing rapid disassembly and reassembly during mitosis and in response to perturbations in membrane trafficking pathways. How Golgi membranes fragment and disperse under these conditions is only beginning to be clarified, but is central to understanding the mechanism(s) underlying Golgi identity and biogenesis. Recent work, discussed in this review, suggests that membrane recycling pathways operating between the Golgi and ER play an indispensable role in Golgi maintenance and biogenesis, with the Golgi dispersing and reforming through the intermediary of the ER both in mitosis and in interphase when membrane cycling pathways are disrupted.     

Golgi membranes are absorbed into and reemerge from the ER during mitosis

Quantitative imaging and photobleaching were used to measure ER/Golgi recycling of GFP-tagged Golgi proteins in interphase cells and to monitor the dissolution and reformation of the Golgi during mitosis. In interphase, recycling occurred every 1.5 hr, and blocking ER egress trapped cycling Golgi enzymes in the ER with loss of Golgi structure. In mitosis, when ER export stops, Golgi proteins redistributed into the ER as shown by quantitative imaging in vivo and immuno-EM. Comparison of the mobilities of Golgi proteins and lipids ruled out the persistence of a separate mitotic Golgi vesicle population and supported the idea that all Golgi components are absorbed into the ER. Moreover, reassembly of the Golgi complex after mitosis failed to occur when ER export was blocked. These results demonstrate that in mitosis the Golgi disperses and reforms through the intermediary of the ER, exploiting constitutive recycling pathways. They thus define a novel paradigm for Golgi genesis and inheritance.     

The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH(2)-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.     

Membrane targeting of p115 phosphorylation mutants and their effects on Golgi integrity and secretory traffic

The cytosolic phosphoprotein p115 is required for ER to Golgi traffic and for Golgi reassembly after mitosis. In cells, p115 is localized to ER exit sites, ER-Golgi Intermediate Compartment (ERGIC) and the Golgi, and cycles between these compartments. P115 is phosphorylated on serine 942, and this modification appears to control p115 association with membranes. P115 is likely to function by reversibly interacting with effector proteins, and in the Golgi, two proteins, GM130 and giantin, have been shown to bind p115. The GM130-p115 and the giantin-p115 interactions are enhanced by p115 phosphorylation. Phosphorylation appears to be essential for p115 function, since substitutions of serine 942 abolish p115 ability to sustain cisternal reformation in an in vitro assay reconstituting Golgi reassembly after mitosis. Here, we explored how phosphorylation of p115 affects its intracellular targeting to distinct cellular compartments, and its function in secretory traffic. We generated phosphorylation mutants of p115 and tested their ability to target to ER exit sites, ERGIC and the Golgi. In addition, we explored whether expression of the mutants causes disruption of Golgi structure and perturbs ER-Golgi traffic of a VSV-G cargo protein.     

Molecular mechanism of mitotic Golgi disassembly and reassembly revealed by a defined reconstitution assay

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.     

Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway

NSF and p97 are related AAA proteins implicated in membrane trafficking and organelle biogenesis. p97 is also involved in pathways that lead to ubiquitin-dependent proteolysis, including ER-associated degradation (ERAD). In this study, we have used dominant interfering ATP-hydrolysis deficient mutants (NSF(E329Q) and p97(E578Q)) to compare the function of these AAA proteins in the secretory pathway of mammalian cells. Expressing NSF(E329Q) promotes disassembly of Golgi stacks into dispersed vesicular structures. It also rapidly inhibits glycosaminoglycan sulfation, reflecting disruption of intra-Golgi transport. In contrast, expressing p97(E578Q) does not affect Golgi structure or function; glycosaminoglycans are normally sulfated and secreted, as is the VSV-G ts045 protein. Instead, expression of p97(E578Q) causes ubiquitinated proteins to accumulate on ER membranes and slows degradation of the ERAD substrate cystic-fibrosis transmembrane-conductance regulator. In addition, expression of p97(E578Q) eventually causes the ER to swell. More specific assessment of effects of p97(E578Q) on organelle assembly shows that the Golgi apparatus disperses and reassembles normally after treatment with brefeldin A and during mitosis. These findings demonstrate that ATP-hydrolysis-dependent activities of NSF and p97 in the cell are not equivalent and suggest that only NSF is directly involved in regulating membrane fusion.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.