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Hisao Kondo

Publications and source records attributed to Hisao Kondo.

16 recordsLinked to original sources
PubMed2006

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.

Adenosine Triphosphatases↗
PubMed2006

p37 is a p97 adaptor required for Golgi and ER biogenesis in interphase and at the end of mitosis.

We previously reported that p97/p47-assisted membrane fusion is important for the reassembly of organelles at the end of mitosis, but not for their maintenance during interphase. We have now identified a p97 adaptor protein, p37, which forms a complex with p97 in the cytosol and localizes to the Golgi and ER. siRNA experiments revealed that p37 is required for Golgi and ER biogenesis. Injection of anti-p37 antibodies into cells at different cell cycle stages showed that p37 plays an important role in both Golgi and ER maintenance during interphase as well as in their reassembly at the end of mitosis. In an in vitro Golgi reassembly assay, the p97/p37 complex has membrane fusion activity. In contrast to the p97/p47 pathway, this pathway requires p115-GM130 tethering and SNARE GS15, but not syntaxin5. Interestingly, although VCIP135 is also required, its deubiquitinating activity is unnecessary for p97/p37-mediated activities.

Adaptor Proteins, Signal Transducing↗
PubMed2005

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.

Adenosine Triphosphatases↗
PubMed2005

The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase.

The endoplasmic reticulum (ER) has a characteristic complex polygonal structure with hallmark three-way junctions in many types of cells. To investigate the mechanisms responsible for maintaining the ER network, we established ER disassembly and reassembly assays in semi-intact Chinese hamster ovary (CHO) cells that constitutively expressed heat shock protein-47 fused to the green fluorescent protein (GFP-HSP47) as an ER marker (the cells are referred to as CHO-HSP cells). Using these assays, we found that maintenance of the ER network required cytosol and adenosine triphosphate/guanosine 5'-triphosphate (ATP/GTP) hydrolysis, but not actin filaments or microtubules. We also showed that the ER network was disrupted upon addition of either N-ethylmaleimide-treated cytosol after washing semi-intact cells with high salt solution or mitotic cytosol in nocodazole-treated semi-intact CHO-HSP cells. The disrupted ER network induced by mitotic cytosol was reformed by the addition of interphase cytosol. In addition, we found that p47, a cofactor of p97, was essential for the maintenance of the ER network, and that phosphorylation of p47 by cdc2 kinase resulted in ER network disruption by mitotic cytosol. Taken together, these results imply that the maintenance of the ER network requires a membrane fusion process mediated by p97/p47, and that cell cycle-dependent morphological changes of the ER network are regulated through phosphorylation/dephosphorylation of p47.

Adenosine Triphosphatases↗
PubMed2005

NSF/SNAPs and p97/p47/VCIP135 are sequentially required for cell cycle-dependent reformation of the ER network.

The endoplasmic reticulum (ER) has a characteristic polygonal structure with hallmark three-way junctions. In a previous paper, we reconstituted the disruption of the pre-existing ER network using mitotic cytosol from HeLa cells in streptolysin O (SLO)-permeabilized CHO-HSP cells (stably expressing GFP-HSP47). In addition, we found that interphase cytosol induced reformation of the disrupted ER network into a continuous network structure. Here, we show that the reformation of the ER network is accomplished through two sequential fusion reactions. The first process is mediated by NSF/alpha and gamma-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.

Adenosine Triphosphatases↗
PubMed2004

Cdc2 kinase-dependent disassembly of endoplasmic reticulum (ER) exit sites inhibits ER-to-Golgi vesicular transport during mitosis.

We observed the disassembly of endoplasmic reticulum (ER) exit sites (ERES) by confocal microscopy during mitosis in Chinese hamster ovary (CHO) cells by using Yip1A fused to green fluorescence protein (GFP) as a transmembrane marker of ERES. Photobleaching experiments revealed that Yip1A-GFP, which was restricted to the ERES during interphase, diffused throughout the ER network during mitosis. Next, we reconstituted mitotic disassembly of Yip1A-GFP-labeled ERES in streptolysin O-permeabilized CHO cells by using mitotic L5178Y cytosol. Using the ERES disassembly assay and the anterograde transport assay of GFP-tagged VSVGts045, we demonstrated that the phosphorylation of p47 by Cdc2 kinase regulates the disassembly of ERES and results in the specific inhibition of ER-to-Golgi transport during mitosis.

Animals↗
PubMed2004

Structure, dynamics and interactions of p47, a major adaptor of the AAA ATPase, p97.

p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Structural basis of the interaction between the AAA ATPase p97/VCP and its adaptor protein p47.

The AAA ATPase p97/VCP is involved in many cellular events including ubiquitin-dependent processes and membrane fusion. In the latter, the p97 adaptor protein p47 is of central importance. In order to provide insight into the molecular basis of p97 adaptor binding, we have determined the crystal structure of p97 ND1 domains complexed with p47 C-terminal domain at 2.9 A resolution. The structure reveals that the p47 ubiquitin regulatory X domain (UBX) domain interacts with the p97 N domain via a loop (S3/S4) that is highly conserved in UBX domains, but is absent in ubiquitin, which inserts into a hydrophobic pocket between the two p97 N subdomains. Deletion of this loop and point mutations in the loop significantly reduce p97 binding. This hydrophobic binding site is distinct from the predicted adaptor-binding site for the p97/VCP homologue N-ethylmaleimide sensitive factor (NSF). Together, our data suggest that UBX domains may act as general p97/VCP/CDC48 binding modules and that adaptor binding for NSF and p97 might involve different binding sites. We also propose a classification for ubiquitin-like domains containing or lacking a longer S3/S4 loop.

Adenosine Triphosphatases↗
PubMed2003

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.

Adenosine Triphosphatases↗
PubMed2003

Motions and negative cooperativity between p97 domains revealed by cryo-electron microscopy and quantised elastic deformational model.

p97, a Mg-ATPase belonging to the AAA (ATPase associated with various cellular activities) super family of proteins, has been proposed to function in two distinct cellular pathways, namely homotypic membrane fusion and ubiquitin protein degradation by utilizing differing adaptor complexes. We present the cryo-electron microscopy three-dimensional reconstruction of endogenous p97 in an AMP-PNP bound state at 24 A resolution. It reveals clear nucleotide-dependent differences when compared to our previously published "p97-ADP" reconstruction, including a striking rearrangement of N domains and a positional change of the two ATPase domains, D1 and D2, with respect to each other. The docking of the X-ray structure of N-D1 domains in an ADP bound state indicates that an upward repositioning of N domain is necessary to accommodate the cryo-EM map of "p97-AMP-PNP", suggesting a change in the orientation of N domains upon nucleotide hydrolysis. Furthermore, computational analysis of the deformational motions of p97, performed on the cryo-EM density map and the atomic structure of the N-D1 domains independently, shows the existence of a negative cooperativity between the D1 and D2 rings and the flexibility of the N domains. Together these results allow the identification of functionally important features that offer molecular insights into the dynamics of the proposed p97 chaperone function.

Adenosine Triphosphatases↗
PubMed2003

The crystal structure of murine p97/VCP at 3.6A.

p97/VCP is a member of the AAA ATPase family and has roles in both membrane fusion and ubiquitin dependent protein degradation. Here, we present a 3.6A crystal structure of murine p97 in which D2 domain has been modelled as poly-alanine and the remaining approximately 100 residues are absent. The resulting structure illustrates a head-to-tail packing arrangement of the two p97 AAA domains in a natural hexameric state with D1 ADP bound and D2 nucleotide free. The head-to-tail packing arrangement observed in this structure is in contrast to our previously predicted tail-to-tail packing model. The linker between the D1 and D2 domains is partially disordered, suggesting a flexible nature. Normal mode analysis of the crystal structure suggests anti-correlated motions and distinct conformational states of the two AAA domains.

Adenosine Triphosphatases↗
PubMed2002

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.

Adenosine Triphosphatases↗
PubMed2002

Involvement of the ryanodine-sensitive Ca2+ store in GLP-1-induced Ca2+ oscillations in insulin-secreting HIT cells.

We investigated intracellular Ca(2+) ([Ca(2+)](i)) oscillations evoked by glucagon-like peptide 1 (GLP-1) in relation to the ryanodine receptor (RyR) and Ca(2+)-induced Ca(2+)release (CICR) mechanism in pancreatic B cell HIT. GLP-1 produced [Ca(2+)](i) oscillations in the cells, both in media with and without Ca(2+), an effect inhibited by ruthenium red and mimicked by 8-Br-cAMPS. In addition, the GLP-1-evoked [Ca(2+)](i) rise was initiated at the local intercellular peripheral cytoplasm, and a resultant expansion of the intercellular space was also observed. Caffeine induced [Ca(2+)](i) elevation in the medium with or without Ca(2+), an effect inhibited by ruthenium red. GLP-1-evoked [Ca(2+)](i) oscillations were also enhanced by IBMX, and eliminated by Rp-8-Br-cAMPS or 20 microM H-89 treatments whereas they were unaffected by 2 microM H-89 treatment. Forskolin caused a transient elevation in [Ca(2+)](i) that was reduced by Rp-8-Br-cAMPS, 2 microM or 20 microM H-89. Our results indicate that GLP-1 initially generated a local [Ca(2+)](i) elevation at the peripheral cytoplasm, subsequently producing [Ca(2+)](i) oscillations that were inhibited by ruthenium red, involving ryanodine-sensitive and cAMP-activated CICR mechanisms. The cytoplasmic levels of cAMP as well as local Ca(2+) might be responsible for [Ca(2+)](i) oscillations.

Calcium↗
PubMed2002

Crystal structure of archaeosine tRNA-guanine transglycosylase.

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.

Amino Acid Sequence↗