At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. The first such component, a protein of relative molecular mass 47,000 (p47), has now been identified. p47 forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. Since a homolog of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy (Kondo, 1997).
The highly conserved ATPase p97, a member of the AAA-ATPases, is found in a complex with its co-factor p47 in rat liver cytosol. Previously it had been shown that p97-mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47, which mediates the binding of p97 to a Golgi t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment factor receptor), syntaxin 5. p47 also suppresses the ATPase activity of p97 by up to 85% in a dose-dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle (Meyer, 1998).
AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis reveals that a major conformational change of p97 during the ATPase cycle occurs upon nucleotide binding and not during hydrolysis, as previously hypothesized. Furthermore, six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners (Rouiller, 2000).
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. Another binding complex comprising mammalian Ufd1 and Npl4 is reported in this study. 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 Ufd1/Npl4 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 (Meyer, 2000).
p47 is the major protein identified in complex with the cytosolic AAA ATPase p97. It functions as an essential cofactor of p97-regulated membrane fusion, which has been suggested to disassemble t-t-SNARE complexes and prepare them for further rounds of membrane fusion. The high-resolution NMR structure of the C-terminal domain from p47 is reported in this study. It comprises a UBX domain and a 13 residue long structured N-terminal extension. The UBX domain adopts a characteristic ubiquitin fold with a ßßalphaßßalphaß secondary structure arrangement. Three hydrophobic residues from the N-terminal extension pack closely against a cleft in the UBX domain. The p97 interaction surface has been identified using NMR chemical shiftperturbation studies (Yuan, 2001).
Although nuclear envelope (NE) assembly is known to require the GTPase Ran, the membrane fusion machinery involved is uncharacterized. NE assembly involves formation of a reticular network on chromatin, fusion of this network into a closed NE and subsequent expansion. p97, an AAA-ATPase previously implicated in fusion of Golgi and transitional endoplasmic reticulum (ER) membranes together with the adaptor p47, has two discrete functions in NE assembly. Formation of a closed NE requires the p97-Ufd1-Npl4 complex, not previously implicated in membrane fusion. Subsequent NE growth involves a p97-p47 complex. This study provides the first insights into the molecular mechanisms and specificity of fusion events involved in NE formation (Hetzer, 2001).
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