Golgi Membrane Fusion Research Articles

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.

Double prenylation of SNARE protein Ykt6 is required for lysosomal hydrolase trafficking.

Ykt6 is an evolutionarily conserved SNARE protein regulating Golgi membrane fusion and other diverse membrane trafficking pathways. Unlike most SNARE proteins, Ykt6 lacks a transmembrane domain but instead has a tandem cysteine motif at the C-terminus. Recently, we have demonstrated that Ykt6 undergoes double prenylation at the C-terminal two cysteines first by farnesyltransferase and then by a newly identified protein prenyltransferase named geranylgeranyltransferase type-III (GGTase-III). GGTase-III consists of a novel α subunit prenyltransferase alpha subunit repeat containing 1 (PTAR1) and the β subunit of Rab geranylgeranyltransferase. PTAR1 knockout (KO) cells, where Ykt6 is singly prenylated with a farnesyl moiety, exhibit structural and functional abnormalities in the Golgi apparatus with delayed intra-Golgi trafficking and impaired protein glycosylation. It remains unclear whether the second prenylation of Ykt6 is required for proper trafficking of lysosomal hydrolases from Golgi to lysosomes. Here, we show that lysosomal hydrolases, cathepsin D and β-hexosaminidase, were missorted at the trans-Golgi network and secreted into the extracellular space in PTAR1 KO cells. Moreover, maturation of these hydrolases was disturbed. LC3B, an autophagy marker, was accumulated in PTAR1 KO cells, suggesting defects in cellular degradation pathways. Thus, doubly prenylated Ykt6, but not singly prenylated Ykt6, is critical for the efficient sorting and trafficking of acid hydrolases to lysosomes.

DjA1 maintains Golgi integrity via interaction with GRASP65.

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis.

Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi-cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor-mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain-containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

Mena-GRASP65 interaction couples actin polymerization to Golgi ribbon linking.

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.

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.

Monoubiquitination Regulates Golgi Membrane Dynamics In The Cell Cycle

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post‐mitotic Golgi membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post‐mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97‐mediated Golgi membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi‐localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced HACE1 activity is human cancer such as Wilms' tumor results in fragmented Golgi. We have also discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post‐mitotic Golgi reassembly. VCIP135 activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and activated upon dephosphorylation in telophase. Recently, we have identified the substrates on the Golgi membranes whose ubiquitination and deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division.

Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

The assembly conformation and kinetics of phosphatidylethanolamine (PE) lipids are the key to their membrane curvatures and activities, such as exocytosis, endocytosis and Golgi membrane fusion. In the current study, a polarization and frequency resolved (bandwidth ≈ 1 cm(-1)) picosecond sum frequency generation (SFG) system was developed to characterize phosphatidylethanolamine monolayers. In addition to obtaining π-A isotherms and Brewster angle microscopy (BAM) images, the conformational changes and assembly behaviors of phosphatidylethanolamine molecules are investigated by analyzing the SFG spectra collected at various surface pressures (SPs). The compression kinetics and relaxation kinetics of phosphatidylethanolamine monolayers are also reported. The conformational changes of PE molecules during the monolayer compression are separated into several stages: reorientation of the head group PO2(-) in the beginning of the liquid-expanded (LE) phase, conformational changes of head group alkyl chains in the LE phase, and conformational changes of tail group alkyl chains in the LE-liquid condensed (LE-LC) phase. Such an understanding may help researchers to effectively control the lipid molecular conformation and membrane curvatures during the exocytosis/endocytosis processes.

The biology of A20-like molecules.

A20 is a ubiquitin-editing molecule. It belongs to a novel family of deubiquitinating cysteine proteases, called the ovarian tumor (OTU) family, which can cleave monoubiquitin from modified proteins. In addition, A20 contains seven Cys2-Cys2 zinc fingers, one of which is believed to regulate E3 ubiquitin ligase activity. Here we review the biology of human genes that encode OTU domains or contain A20-type zinc fingers. The human genome contains 15 members of the OTU family including the deubiquitinating enzymes Cezanne, VCIP135 and Otubain 1. Genomic analysis also identified 10 genes that contain A20-type zinc fingers including Rabex5, Znf216 and AWP1. In Rabex5 the A20-zinc finger regulates E3 ligase activity whereas A20-type zinc fingers of Znf216 and AWP1 function as ubiquitin-binding motifs. A20 and its relatives regulate highly divergent physiological activities including NF-kappaB activity (A20, Cezanne, Znf216, Rabex5), endocytosis (Rabex5, AWP1), skeletal muscle atrophy (Znf216), Golgi membrane fusion (VCIP135) and T-cell anergy (Otubain 1). Further studies are required to characterize the biology of other A20-related molecules whose function remains largely undefined.

Abstract 2146: Development of functional assays for p97/VCP inhibition.

Abstract p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system. p97 also plays a critical role in specific cellular processes including Golgi membrane reassembly, membrane fusion, autophagy and DNA repair mechanisms. p97 expression is elevated in a number of cancer types including prostate cancer, non-small-cell lung carcinoma and leukemia. Given its cellular functions in protein homeostasis and the successful clinical development of proteasome inhibitors, p97 has emerged as an exciting new target for cancer therapy. We used p97 siRNA and a recently identified small molecule p97 inhibitor, N2,N4-dibenzylquinazoline- 2,4-diamine (DBeQ) as tools to develop functional assays suitable for further evaluating the activity of potential p97 inhibitors. A panel of unfolded protein response marker genes that are consistently modulated by p97 siRNA or DBeQ treatment has been identified. In addition, methods to monitor autophagic activity have been developed, including high content analysis of LC3B-GFP puncta formation. Cell viability assays were used to determine the susceptibility of various cancer cell lines to p97 inhibition. These assays will be used to evaluate the effects of potential p97 inhibitors on p97-dependent pathways and anti-proliferative activity. Acknowledgement: Funded by NExT Chemical Biology Consortium, NCI Contract No. HHSN261200800001E. Citation Format: Yihui Shi, Xiaohe Liu, Joy Calaoagan, Steven Samuelsson, Tsui-Fen Chou, Raymond J. Deshaies, Lidia C. Sambucetti. Development of functional assays for p97/VCP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2146. doi:10.1158/1538-7445.AM2013-2146

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

Cell cycle regulation of Golgi membrane dynamics

The Golgi apparatus is a membranous organelle in the cell that plays essential roles in protein and lipid trafficking, sorting, processing, and modification. Its basic structure is a stack of closely aligned flattened cisternae. In mammalian cells, dozens of Golgi stacks are often laterally linked into a ribbon-like structure. Biogenesis of the Golgi during cell division occurs through a sophisticated disassembly and reassembly process that can be divided into three distinct but cooperative steps, including the deformation and reformation of the Golgi cisternae, stacks, and ribbon. Here, we review our current understanding of the protein machineries that control these three steps in the cycle of mammalian cell division: GRASP65 and GRASP55 in Golgi stack and ribbon formation; ubiquitin and AAA ATPases in postmitotic Golgi membrane fusion; and golgins and cytoskeleton in Golgi ribbon formation.

Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97-p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135 function requires membrane association and p97 interaction, both of which are inhibited in mitosis by VCIP135 phosphorylation. We found that wild-type VCIP135, but not its phosphomimetic mutants, rescues Golgi structure in VCIP135-depleted cells. Our results demonstrate that VCIP135 phosphorylation regulates its Golgi membrane association and p97 interaction, and thus contributes to the tight control of the Golgi disassembly and reassembly process during the cell cycle.

P47 negatively regulates IKK activation by inducing the lysosomal degradation of polyubiquitinated NEMO

The persistent or excess activation of NF-κB causes various inflammatory and autoimmune diseases, but the molecular mechanisms that negatively regulate NF-κB activation are not fully understood. Here we show that p47, an essential factor for Golgi membrane fusion, associates with the NEMO subunit of the IκB kinase (IKK) complex upon TNF-α or IL-1 stimulation, and inhibits IKK activation. p47 binds to Lys63-linked and linear polyubiquitin chains, which are conjugated to NEMO upon such stimulation. The binding of p47 to polyubiquitinated NEMO triggers the lysosomal degradation of NEMO, thereby inhibiting IKK activation. The silencing of p47 results in enhanced TNF-α- or IL-1-induced IKK activation, and an increased expression of genes encoding inflammatory mediators. Taken together, our results suggest that p47 is critical for negatively regulating stimulation-induced IKK activation in a manner that is mechanistically distinct from the previously characterized negative regulators, such as A20 and CYLD.

The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.

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.

Isolation of a point-mutated p47 lacking binding affinity to p97ATPase

p47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97. Structured summary MINT- 7987189, MINT- 7987207, MINT- 7987303: p47 (uniprotkb: O35987) binds (MI: 0407) to p97 (uniprotkb: Q01853) by pull down (MI: 0096) MINT- 7987226: p97 (uniprotkb: P46462) binds (MI: 0407) to p47 (uniprotkb: O35987) by pull down (MI: 0096) MINT- 7987348: p97 (uniprotkb: P46462) physically interacts (MI: 0915) with Ufd1 (uniprotkb: P70362) by pull down (MI: 0096) MINT- 7987264: p97 (uniprotkb: P46462) and p47 (uniprotkb: O35987) bind (MI: 0407) by competition binding (MI: 0405) MINT- 7987326: p97 (uniprotkb: P46462) binds (MI: 0407) to p37 (uniprotkb: Q0KL01) by pull down (MI: 0096)

Temporal Global Expression Data Reveal Known and Novel Salicylate-Impacted Processes and Regulators Mediating Powdery Mildew Growth and Reproduction on Arabidopsis

Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wild-type and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T(2) statistic). Functional analyses of T(2)-selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.

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.

The Role of a Novel p97/Valosin-containing Protein-interacting Motif of gp78 in Endoplasmic Reticulum-associated Degradation

Improperly folded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation, a process that dislocates misfolded proteins from the ER membrane into the cytosol, where they undergo proteasomal degradation. Dislocation requires a subclass of ubiquitin ligases that includes gp78 in addition to the AAA ATPase p97/VCP and its cofactor, the Ufd1-Npl4 dimer. We have previously reported that gp78 interacts directly with p97/VCP. Here, we identify a novel p97/VCP-interacting motif (VIM) within gp78 that mediates this interaction. We demonstrate that the VIM of gp78 recruits p97/VCP to the ER, but has no effect on Ufd1 localization. We also show that gp78 VIM interacts with the ND1 domain of p97/VCP that was shown previously to be the binding site for Ufd1. To evaluate the role of Ufd1 in gp78-p97/VCP-mediated degradation of CD3delta, a known substrate of gp78, RNA interference was used to silence the expression of Ufd1 and p97/VCP. Inhibition of p97/VCP, but not Ufd1, stabilized CD3delta in cells that overexpress gp78. However, both p97/VCP and Ufd1 appear to be required for CD3delta degradation in cells expressing physiological levels of gp78. These results raise the possibility that Ufd1 and gp78 may bind p97/VCP in a mutually exclusive manner and suggest that gp78 might act in a Ufd1-independent degradation pathway for misfolded ER proteins, which operates in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.