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1 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan
2 Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 2XY, UK s
3 Mitsubishi Institute of Life Science, Machida, Tokyo 194-8511, Japan
4 PRESTO, Japan Science and Technology Corporation, Japan
5 Cellular Structure Laboratory, Cell Biology Program, Nagahama Institute of Bioscience and Technology, Shiga 526-0829, Japan
6 Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
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Abstract |
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-SNAPs, and involves the generation of typical membranous intermediate structures that connect the disrupted ER tubules. A subsequent fusion is mediated by p97/p47/VCIP135, which has been shown to be required for homotypic fusion events in Golgi cisternae regrowth after mitosis. In addition, we also found that both fusion processes involve the t-SNARE, syntaxin 18. |
Introduction |
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Dreier and Rapoport (2000) reconstituted ER network formation from microsomal membrane vesicles prepared from Xenopus laevis eggs in vitro. They showed that the formation of both the ER network and the nuclear envelope occurs in interphase extracts, but not in cdc2 kinase-activated interphase extracts, which mimic mitotic conditions. They also found that N-ethylmaleimide (NEM)-sensitive factors associated with the membranes, ATP, and other cytosolic factor(s) are required for tubule and ER network formation. Remarkably, the process was found to be independent of microtubule integrity.
Hetzer et al. (2001) reported that an NEM-sensitive AAA ATPase, p97 (Cdc48p in yeast), together with the adaptor protein p47, has two discrete functions in nuclear envelope assembly and is a component of the fusion machinery forming three-way junctions in the ER network. p97 has been reported to induce the homotypic fusion of ER membranes during yeast mating (Latterich et al. 1995; Patel et al. 1998) of vesiculated Golgi membranes at mitosis (Rabouille et al. 1995) and of ilimaquinone-induced Golgi fragments (Acharya et al. 1995). The protein is also involved in the formation of transitional ER, which is a specific region of the ER where cargo and coat proteins are concentrated (Roy et al. 2000; Kano et al. 2004), extraction of polyubiquitinated proteins from the ER to cytosol (Ye et al. 2001), and spindle pole disassembly (Cao et al. 2003). In contrast, an appropriate experimental system for studying the deformation/reformation of the ‘pre-existing’ ER network and three-way junctions has yet to be established.
More recently, we produced the stable transfectant, CHO-HSP, which continuously expresses heat shock protein-47 fused to GFP (GFP-HSP47). We have examined the biochemical requirements for morphological changes of the pre-existing ER network in semi-intact CHO-HSP cells (Kano et al. 2005). Semi-intact cells are cells whose plasma membranes have been permeabilized by detergents or toxins, and which have been used for our reconstitution of the mitotic Golgi fragmentation (Kano et al. 2000a), brefeldin A-induced Golgi tubulation and fusion with the ER (Kano et al. 2000b), and the mitotic disruption of ER exit sites (Kano et al. 2004). Based on this assay, we found that maintenance of the network is mediated by NEM-sensitive factors that are tightly associated with the membrane. The network was severed in a cdc2 kinase-dependent manner by mitotic cytosol from HeLa cells. In addition, we found that the specific phosphorylation of p47 by cdc2 kinase is a trigger for mitotic disruption in semi-intact cells. To our surprise, lumenal continuity is maintained in mitotic cytosol-treated semi-intact cells despite the fact that the ER network is partially severed. Ellenberg et al. (1997) and Terasaki (2000) also demonstrated that the ER network remains interconnected in COS-7 cells or in sea urchin embryos during mitosis, by using GFP-tagged lamin B receptors or KDEL-receptors for visualization of the network, together with photobleaching techniques. The structural features of ER tubules severed by mitotic cytosol were indistinguishable from those resulting from the loss of function of NEM-sensitive factors. These results indicated a possible role for the p97/p47 complex and/or NSF in the maintenance (or fusion between ER tubules) of the network. In that paper (Kano et al. 2005) we used interphase HeLa cytosol to reconstitute the reformation of the ER network of semi-intact CHO-HSP cells, which had been previously disrupted by incubation with mitotic cytosol.
In the present study using this reconstitution system, we found that ER network reformation is not accomplished by a single fusion reaction mediated by p97/p47 complexes, but rather requires two sequential fusion reactions. The first fusion event is mediated by NSF/ and -SNAPs, and involves the generation of fine junctions and pleiomorphic vesicle aggregates between the disrupted ER tubules. In the second process, p97/p47/VCIP135, which is the minimal cytosolic protein component required for the homotypic membrane fusion of vesiculated Golgi following mitosis, induces the fusion of connected ER tubules to form three-way junctions. In addition, we also found that both processes require the t-SNARE, syntaxin 18.
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Results |
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In a previous paper (Kano et al. 2005), we found that mitotic cytosol disrupted the ER network in semi-intact CHO-HSP cells and that this disruption was regulated by cdc2 kinase and its substrate p47, which is a cofactor of p97. In addition, the disrupted network reformed a continuous network structure following incubation with interphase cytosol/ATP, but microtubules were not required for the reformation.
We have monitored the dynamics of ER tubules during reformation using time-lapse fluorescence microscopy under high magnification. We found that the disrupted ER tubules bifurcated and elongated under every experimental condition: in the presence of either mitotic or interphase cytosol, or even in the presence only of a buffer containing an ATP-regenerating system. Hence, we hypothesized that membrane fusion but not tubulation/bifurcation could be a crucial step for ER network reformation, and that general fusion proteins such as NSF or p97 are involved in reformation.
To test this, we prepared NEM-treated interphase (NEM(I)) cytosol, in which ATPases such as NSF and p97 were inactivated, and examined its effect on reformation. CHO-HSP cells, which stably express GFP-tagged HSP47 as a luminal marker of the ER, were permeabilized with streptolysin O (SLO), and then incubated with mitotic cytosol, which causes ER disruption. After washing out mitotic cytosol, we added interphase- or NEM-treated cytosol into the semi-intact cells. We found that incubation with either interphase or NEM(I) cytosol permitted ER network reformation (Fig. 1A, a and b, and Fig. 1B, M
I and MNEM(I)). However, when we washed the semi-intact cells extensively with 2 M KCl after disruption of the ER network, three-way junctions were not reformed by addition of NEM(I) cytosol (Figs 1A, c and d, Fig. 1B, M2 M KCl washI, and M2 M KCl washNEM(I)). In fact, the high-salt wash released > 90% of membrane-associated NSF or p97, which was confirmed by Western blotting (data not shown).
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To investigate further the involvement of NSF and p97 in ER network reformation, we studied the effects of antibodies against NSF and p47, which forms a tight stoichiometric complex with p97 (Kondo et al. 1997) on ER network reformation (Fig. 2A). A polyclonal antibody specific for NSF, which inhibits the fusion of BFA-derived Golgi tubules with the ER (F. Kano, unpublished data), and an anti-p47 antibody inhibited reformation (Fig. 2A, anti-NSF and anti-p47). These data show that NSF and the p97/p47 complex are involved in ER network reformation. Using recombinant proteins, we further examined the requirement for NSF and p97 during reformation.
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-SNAP, 3 µg/mL; -SNAP, 3.4 µg/mL), NEM(I) cytosol and p97s (p97, 20 µg/mL; p47, 22 µg/mL), or NSFs and p97s, ER network reformation was not restored (Fig. 2B, NEM(I)+ NSFs, NEM(I)+p97s, and NSFs+p97s). In contrast, simultaneous addition of NEM(I) with NSFs and p97s facilitated reformation (Fig. 2B, NEM(I)+p97s+NSFs). These results suggested that NSFs, p97s, and other factor(s) in NEM(I) cytosol might be necessary for reformation. Recently, a novel VCIP135 protein has been identified as a p97/p47/SNARE complex recycling factor and was found to be essential for cisternae regrowth in Golgi reassembly after mitosis (Uchiyama et al. 2002). We therefore supplied purified recombinant VCIP135 (6.0 µg/mL) to the mixture of NSFs and p97s, and then performed the reformation assay. Surprisingly, the mixture supported complete ER network reformation (Fig. 2B, NSFs+p97s+VCIP135). In addition, an antibody against VCIP135 inhibited ER reformation (Uchiyama et al. 2002). On the other hand, p115, which plays a role in tethering Golgi-derived vesicles to Golgi cisternae, was not necessary for ER network reformation (data not shown).
Next, we tested the sequential effect of NSFs and p97s+VCIP135 on reformation. After incubation with NSFs (or p97s+VCIP135), the cells were washed with 2 M KCl to release the membrane-bound NSFs (or p97s). The cells were then further incubated with p97s+VCIP135 (or with NSFs). Antibodies to NSFs (or to p47) were added to the second incubation mixtures for complete inactivation of residual NSF (or p97) in 2 M KCl-washed cells. The NSFsp97s+VCIP135 incubation induced full reformation (Fig. 2B, NSFsp97s+VCIP135+anti-NSF). In contrast, the reformation did not occur in the p97s+VCIP135NSFs incubation mixture (Fig. 2B, p97s+VCIP135NSFs+anti-p47). These results suggest that the reformation process consists of sequential fusion reactions: the first step is mediated by NSFs and the second by p97s+VCIP135.
Membranous intermediate structures in the ER network reformation process are created by NSFs
Morphological changes of the ER during reformation mediated by NSFsp97s+ VCIP135 were monitored in a single semi-intact cell using time-lapse fluorescence microscopy (Fig. 3A. Also see supplemental data 1). The semi-intact cell with a disrupted ER network was first incubated with NSFs at 32 °C for 20 min, followed by p97s+VCIP135 to initiate reformation. At an early stage of the reaction, the disrupted ER tubules appeared to merge to form longer tubules (Fig. 3A, and Fig. 3B arrowheads). The merged tubules, which exhibited elastic movements, were connected to side tubules by tubulation/bifurcation, resulting in the formation of three-way junctions (Fig. 3A, and Fig. 3B arrows). As the reformation of the three-way junctions proceeded (Fig. 3A, from 2'37 to 6'08), the ER network occasionally contracted. In the presence of NSFs alone, we observed that the disrupted tubules occasionally appeared to form linear (beads-on-string) arrays.
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Next we examined the fine structural features of the ER during the process of reformation by electron microscopy. In mitotic cytosol-treated cells, disrupted ER tubules were observed throughout the cytoplasm (Fig. 4Ba). When mitotic cytosol-treated cells were incubated with NSFs, fine junctions connecting two individual tubules (Fig. 4B,b and c, arrowheads) or vesicle aggregates (Fig. 4B,b and c, arrows) were frequently observed between the disrupted tubular structures. The size of vesicle aggregates was 
< 1.0 x 1.0 µm2 and the diameter and length of fine junctions was 20–30 nm and < 500 nm, respectively. The vesicle aggregates appeared to be connected to the disrupted ER tubules by fine junctions (Fig. 4B,b and c, arrowheads) and sometimes were organized in a line along the short microtubule fragments (Fig. 4B,b, small arrows). These prominent structures were not seen in the presence of p97s+VCIP135 or NSFs+p97s+VCIP135 (data not shown). Instead, in the presence of NSFs+p97s +VCIP135, we frequently observed long tubular structures with bifurcations (three-way junctions), which were also seen in intact cells (data not shown. For reference, see Fig. 4Bd asterisk). Although we could not ascertain whether the intermediate structures detected by EM were identical to the bright spots labeled with BODIPY TR-ceramide under light microscopy, the morphological data strongly suggest that these typical structures might correspond to intermediate structures during the ER network reformation process.
If the fine junctions or vesicle aggregates described previously are the intermediate structures of the reformation process, these characteristic structures should disappear during the second incubation with p97s+ VCIP135. Hence, after the mitotic cytosol-treated semi-intact cells were incubated with NSFs for 40 min, the cells were further incubated with p97s+VCIP135 and then examined by electron microscopy. Fine junctions and vesicle aggregates largely disappeared within 5 min of the second incubation (Fig. 4Bd), and we sometimes observed vesicle aggregates that appeared to be fusing with disrupted ER tubules (Fig. 4Bd inset, arrow). This was confirmed by the reformation assay using anti-p47 antibodies and interphase cytosol. If p97s was responsible for the disappearance of intermediate structures, the latter would have been observed following inhibition of the second process mediated by the p97/p47 pathway. As shown in Fig. 4Be, similar structures to those seen in cells treated with NSFs were indeed observed.
Why were these fine structures not visible by light microscopy? Indeed, immuno-EM studies using anti-GFP antibody revealed that intermediates of the vesicle aggregates did not contain the GFP-HSP47 (data not shown).
Taken together, we conclude that fine junctions and vesicle aggregates are intermediate structures in the ER network reformation process and that these structures are most likely fusion products facilitated by NSFs. These morphological observations confirmed the sequential fusion reaction mediated by NSFs and p97s.
t-SNARE for NSFs and p97s in the ER network reformation process
To determine which SNAREs are involved in the reformation process, we examined the effect of antibodies against syntaxins 5 and 18, which are known to localize to the ER. A polyclonal antibody against syntaxin 5, which can inhibit the fusion of mitotic vesiculated Golgi membranes in vitro (data not shown), had no effect on ER network reformation (Fig. 5A, anti-syn5). In contrast, an antibody to syntaxin 18 substantially prevented reformation (Fig. 5A, anti-syn18). We confirmed the monospecificity of an anti-syntaxin 18 antibody by immunoblotting (Fig. 5B). Additionally, syntaxin 18 lacking the transmembrane region (syn18(–TM)), competitively inhibited this process (Fig. 5A, syn18(–TM) pre). However, the long form of syntaxin 5 lacking the transmembrane region (syn5(–TM)), had no effect on reformation (data not shown).
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Discussion |
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Biochemical requirements for the reformation of the ER network
The major finding reported in a previous paper was that the disrupted ER tubules, which were induced by mitotic cytosol, reformed a network structure with almost the same shape and size as that in intact cells, by interphase cytosol (Kano et al. 2005). Here, using our reformation assay, we found that the process required only NSFs and p97s+VCIP135 as exogenous factors (Fig. 2B). It was surprising that two fusion processes alone, induced by NSFs and p97s+VCIP135, were sufficient to achieve three-way junction formation in our system, because it has previously been reported that a cytosol-dependent tubulation/bifurcation process, in addition to the vesicle fusion process, was necessary for three-way junction formation (Dreier & Rapoport 2000). Why are exogenous factors for the tubulation/bifurcation process unnecessary? Our light microscopic observations revealed that tubulation/bifurcation of the disrupted ER tubules occurred even when semi-intact cells were incubated with mitotic cytosol or ATP/TB buffer only. We therefore assumed that tubulation/bifurcation might be a property of the ER membrane itself or the residual proteins in semi-intact cells might be sufficient for this process.
During the reformation assay, we did not detect substantial reconstruction of microtubules at the light microscopic level. This indicated that the integrity of microtubule networks is not necessary for reformation of the ER network, which is consistent with the report by Dreier and Rapoport (2000). However, we found some short microtubule fragments associated with the disrupted ER tubules in our EM study (Fig. 4B). Moreover, some investigators reported that the formation of ER networks was microtubule-dependent (Dabora & Sheetz 1988; Allan 1998). Hence, we cannot rule out the possibility that the microtubule fragments helped the disrupted ER tubules to be incorporated into the networks.
ER network reformation by sequential fusion reactions induced by NSF/SNAPs and p97/p47/VCIP135
Our results demonstrated that NSFs are necessary for ER network reformation. However, Hetzer et al. (2001) showed that the p97/p47 complex suffices for ER network formation but NSF does not. This discrepancy may be the result of differences in the membranes used for the ER network reformation assay. Hetzer et al. (2001) used membrane vesicle fractions derived from Xenopus eggs without a high-salt wash. Instead, we used the high-salt washed, pre-existing ER tubular membranes in semi-intact cells. By washing, residual NSF, which is tightly membrane-associated, was depleted and its effect on the early steps of ER tubule fusion might thereby be more effectively revealed.
Our ER network reformation assay showed that syntaxin 18 is involved in fusion reactions mediated both by NSF and p97. Syntaxin 18 was identified as an -SNAP-associating protein and was found to localize principally to ER membranes. It has been proposed that syntaxin 18 is involved in NSF-dependent fusion in the vesicular traffic between the ER and the Golgi, and that its over-expression causes remarkable aggregation of ER membranes. Moreover, several structural and functional features of syntaxin 18 are similar to those of Ufe1p (Hatsuzawa et al. 2000), which mediates ER membrane fusion in yeast karyogamy (Patel et al. 1998).
We demonstrated the binding of syntaxin 18 to p97 via p47. Syntaxin 18 contains two predicted coiled-coil regions, which might cause nonspecific binding of p47 to syntaxin 18 in vitro. We considered that p47 did not bind to coiled-coil proteins nonspecifically because p47 were shown to interact with syntaxin 5 (Rabouille et al. 1998), but not with another protein that contains coiled-coil regions such as small VCP/p97 interacting protein (SVIP) (Nagahama et al. 2003) in binding assay with purified recombinant proteins. This indicates that binding of p47 to syntaxin 18 was specific.
In the Golgi reassembly process from mitotic Golgi fragments, NSF/-SNAP and p97/p47 share a common receptor, syntaxin 5, and competitively bind to it. As a result, the NSF pathway, which generates fenestrated cisternae, was inhibited by p47, and the p97 pathway, which generates unfenestrated cisternae, was suppressed by -SNAP (Rabouille et al. 1998). It is open to question how NSF and p97 correctly use this common t-SNARE during ER network reformation. Interestingly, the competitive inhibition of excess -SNAP was not observed in ER network reformation (data not shown). Because Ufe1p, a yeast homolog of syntaxin 18, is reported to show several polymerization forms (Patel et al. 1998), one possible explanation is that there are several forms of syntaxin 18. These might have different localizations in mitotic ER membranes and/or distinct binding affinities for NSF/SNAPs and p97/p47.
We found that NSFs and p97s act sequentially in the reformation process. The first step is mediated by NSFs and the second by p97s+VCIP135 (Fig. 2). In the case of the Golgi reassembly process from mitotic fragments or drug-induced vesicles, both NSFs and p97s were reported to be essential. However, the effects of NSF and p97 on Golgi reassembly were parallel (Rabouille et al. 1995) or additive (Acharya et al. 1995). What does this imply for the sequential fusion reactions in the ER network? An attractive answer could be provided by our finding that NSFs generate typical membranous structures as described below.
Potential mechanisms of ER network reformation by NSFs and p97s
Figure 6 shows our working model of ER network reformation. The ER network is transiently severed under mitotic conditions in a cdc2-dependent manner. Electron microscopic observations showed that NSFs could generate typical membranous structures, fine junctions, and vesicle aggregates between the disrupted ER tubules during the ER network reformation process (Fig. 4B,b and c). These intermediate structures were also observed following addition of anti-p47 antibody together with interphase cytosol (Fig. 4Be). Our light microscopy observations also revealed that the vesicle aggregates, which originally might be derived from GFP-HSP47-negative membranes, were membranous products induced by NSFs-mediated fusion processes and represent the intermediates of ER network reformation (Fig. 4A). Although we could not ascertain whether the intermediate structures detected by EM were identical to the bright spots labeled with BODIPY TR-ceramide under light microscopy, these data strongly indicate that fine junctions and vesicle aggregates are intermediate structures for ER network reformation. We believe that their function might be to serve as a membranous ‘connecting’ system to facilitate effective second fusion events. Because ER tubules are much larger than the transport vesicles, tight connection of ER tubules by membranous intermediates might be necessary for their fusion.
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Several interesting problems remain to be addressed. First, because the ER network is not uniform, different fusion machinery may be required for the formation of each subset of the ER network. It has been reported that assembly of smooth and rough ER in vitro are differentially affected by non-hydrolysable GTP analogs and addition of anti-p97 antibody (Lavoie et al. 1996; Roy et al. 2000). Second, what kind of molecules affects the NSF functions during mitosis? Considering that no intermediate structures were detected in mitotic cytosol-treated cells (Fig. 4Ba), the first process mediated by NSF appeared to be inhibited during ER disruption. In Golgi disassembly, phosphorylation of GM130 by cdc2 prevents its binding to p115, a vesicle-tethering protein, resulting in the inhibition of NSF-dependent fusion events (Lowe et al. 1998). Although ER network disruption was cdc2-dependent, p115 was not required for ER reformation. Therefore, the first process of ER reformation mediated by NSFs might require an unidentified tethering protein, which is regulated by cdc2. Alternatively, the functions of NSF/SNAPs/SNARE might be directly modified in a cell-cycle dependent manner. More sophisticated reconstitution assays based on our assay will allow us to find the mechanisms and molecular basis of reformation/deformation of the ER network.
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Experimental procedures |
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Nocodazole and N-ethylmaleimide (NEM) were purchased from Sigma. Butyrolactone I (BL, Affiniti Research Products) was stored at 100 mg/mL in DMSO. BODIPYTMTR ceramide was purchased from Molecular Probes, Inc. (Eugene, OR). All other chemicals were from Wako Pure Chemical Industries. The following antibodies were used: rabbit mouse anti--tubulin antibody (Sigma Chemicals Co.); mouse anti-cdc2 antibody (Santa Cruz Biotechnology); and rat antiprotein disulfide isomerase (PDI) antibody from Dr Ryuichi Masaki (Kansai Medical University). Anti-NSF polyclonal antibody was raised against a synthetic peptide (18 amino acids) derived from the 199Ala-216Glu sequence of the NSF protein of CHO cells. Anti-VCP (p97) mouse monoclonal antibody was purchased from Abcam (Cambridgeshire, UK). All other antibodies, the proteins, and the HeLa cytosols were prepared as described in Uchiyama et al. (2002, 2003). CHO-HSP cells, which continuously express GFP-tagged HSP47 (heat shock protein 47), were established and maintained as described in Kano et al. (2005).
Fluorescence and electron microscopy
Samples were examined under a 100x Plan-NEOFLUAR oil immersion objective on a Zeiss confocal microscope LSM 510 equipped with a temperature-controlled chamber. Time-lapse sequences of images were acquired every 2–3 s, and were processed by NIH image software (Wayne Rasband Analytics, NIH).
For electron microscopy, samples were fixed with 2.5% glutaraldehyde at room temperature for 2 h, and processed according to Hatsuzawa et al. (2000).
The ER reformation assay
The ER tubular networks in semi-intact CHO-HSP cells were disrupted using mitotic cytosol as previously described (Kano et al. 2005). The resulting cells were extensively washed with transport buffer (TB; 25 mM HEPES-KOH (pH 7.4), 115 mM potassium acetate, 2.5 mM MgCl2, 1 mM DTT, 2 mM EGTA) for 20 min on ice, followed by 2 M KCl in TB for 25 min on ice. The cells were further incubated with interphase/mitotic cytosol or mixtures of recombinant proteins containing an ATP-regenerating system (1 mM ATP, 8 mM creatine kinase, 50 µg/mL creatine phosphate), 1 mM GTP, and 1 mg/mL glucose at 32 °C for 80 min. The cells were examined under a confocal microscope or used in the three-way junction assay as described in Kano et al. (2005). Briefly, the number of three-way junctions of ER networks in randomly selected fields of the semi-intact cells with a size of 23 x 23 µm2 (almost one quarter of total cell size) was enumerated. Three independent experiments were performed, and means and standard deviations were calculated (n = 30–50). Zero percent represents the number of three-way junctions when semi-intact cells were incubated with mitotic cytosol, and 100% represents that when the cells were incubated with interphase cytosol.
Preparation of a recombinant syntaxin 18 protein without a transmembrane region and an anti-syntaxin 18 polyclonal antibody
The cDNA of rat syntaxin 18 without a transmembrane region (syn18(–TM)) was PCR amplified from rat liver cDNA library and subcloned into pGEX4T-2 for the production of GST-tagged syn18(–TM). GST-tagged syn18(–TM) was expressed in Escherichia coli and purified with glutathione-beads. An anti-syn18 polyclonal antibody was raised against GST-syn18(–TM) and affinity purified.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: mmurata{at}bio.c.u-tokyo.ac.jp
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References |
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Received: 17 June 2005
Accepted: 10 July 2005
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 M. Puhka, H. Vihinen, M. Joensuu, and E. Jokitalo Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells J. Cell Biol., December 3, 2007; 179(5): 895 - 909. [Abstract] [Full Text] [PDF]
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 D. Gorczyca, J. Ashley, S. Speese, N. Gherbesi, U. Thomas, E. Gundelfinger, L. S. Gramates, and V. Budnik Postsynaptic Membrane Addition Depends on the Discs-Large-Interacting t-SNARE Gtaxin J. Neurosci., January 31, 2007; 27(5): 1033 - 1044. [Abstract] [Full Text] [PDF]
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 E. G. Mimnaugh, W. Xu, M. Vos, X. Yuan, and L. Neckers Endoplasmic Reticulum Vacuolization and Valosin-Containing Protein Relocalization Result from Simultaneous Hsp90 Inhibition by Geldanamycin and Proteasome Inhibition by Velcade Mol. Cancer Res., September 1, 2006; 4(9): 667 - 681. [Abstract] [Full Text] [PDF]
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S. M. Millard and S. A. Wood Riding the DUBway: regulation of protein trafficking by deubiquitylating enzymes J. Cell Biol., May 22, 2006; 173(4): 463 - 468. [Abstract] [Full Text] [PDF]
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