Sequential Involvement of p115, SNAREs, and Rab Proteins in Intra-Golgi Protein Transport

Gmachl, Michael; Wimmer, Christian

doi:10.1074/jbc.m101513200

Cited by 9 publications

(12 citation statements)

References 89 publications

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“…Together these data indicate that much of the ␣-SNAP front face and, in particular, the concave face of the twisted sheet is involved in SNARE complex binding. The extensive binding surface defined here is consistent with the large minimal binding domain predicted by previous studies of truncation mutants (8,(21)(22)(23)(24).…”

Section: Snare Complex Binding By ␣-Snapsupporting

confidence: 74%

“…This EC 50 is nevertheless surprisingly low given that the EC 50 of ␣-SNAP for mediating SNARE complex disassembly is ϳ0.2 M (Table I). 0.2 M ␣-SNAP is closer to what is needed for functional reconstitution of NSF-and SNAP-dependent processes in vitro (24,38,39). That co-precipitation-based assays inherently underestimate affinity may in part explain this discrepancy.…”

Section: Discussionmentioning

confidence: 79%

“…Finally, a glycine mutation at Phe-32 led to only a small decrease in SNARE complex binding. This was unexpected because Phe-32 is the only conserved residue not on the back face among the first 35 amino acids of ␣-SNAP, a region reported to be critical for SNARE complex binding (24).…”

Section: Snare Complex Binding By ␣-Snapmentioning

confidence: 88%

“…Based on deletion studies (8,(21)(22)(23)(24) and electron micrographs in which ␣-SNAP molecules appear to ensheathe the SNARE complex (7), a significant portion of ␣-SNAP is thought to participate in its interaction with the SNARE complex. The structure of the Saccharomyces cerevisiae ␣-SNAP orthologue, Sec17p (25), provides some clues to where the interfaces between SNAPs and SNARE complexes might be.…”

mentioning

confidence: 99%

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Defining the SNARE Complex Binding Surface of α-SNAP

Marz

Lauer

Hanson

2003

Journal of Biological Chemistry

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N-Ethylmaleimide-sensitive factor (NSF) and its adaptor protein ␣-soluble NSF attachment protein (␣-SNAP) sustain membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes that form during membrane fusion. To better understand the role of ␣-SNAP in this process, we used sitedirected mutagenesis to identify residues in ␣-SNAP that interact with SNARE complexes. We find that mutations in charged residues distributed over a concave surface formed by the N-terminal nine ␣-helices of ␣-SNAP affect its ability to bind synaptic SNARE complex and promote its disassembly by NSF. Replacing basic residues on this surface with alanines reduced SNARE complex binding and disassembly, whereas replacing acidic residues with alanines enhanced ␣-SNAP efficacy in both assays. These findings show that the ability of NSF to take apart SNARE complexes depends upon electrostatic interactions between ␣-SNAP and the acidic surface of the SNARE complex and provide insight into how NSF and ␣-SNAP work together to drive disassembly.Intercompartmental transport and exocytosis depend upon membrane fusion. This fusion requires membrane-associated helical proteins known as soluble N-ethylmaleimide-sensitive factor (NSF) 1 attachment protein receptors (SNAREs), which coil together into SNARE complexes that are thought to bring membranes together and promote membrane fusion (1-3). After fusion, SNARE complexes must be disassembled to regenerate individual SNAREs for use in subsequent fusion reactions. Proteins responsible for this disassembly are the ATPase NSF and its adaptor protein soluble NSF attachment protein (SNAP). Insight into this process is important to understanding how membrane trafficking is controlled in the cell.␣-SNAP (a ubiquitous SNAP isoform) serves as the requisite link between NSF and SNARE complexes. It binds to SNARE complexes, and together they recruit NSF (4). SNARE-bound ␣-SNAP stimulates ATP hydrolysis by NSF, leading to conformational changes and concomitant disassembly of the SNARE complex (2, 4). Scanning transmission electron microscopy has been used to define the probable composition of the complex containing ␣-SNAP, NSF, and SNARE complex, also referred to as 20 S complex (5). Each 20 S complex consists of one SNARE complex, three ␣-SNAPs, and one NSF hexamer. Interestingly, ␣-SNAP only binds NSF in the absence of SNARE complex when forced into an oligomeric form by fusion to an intrinsically trimeric protein (5) or when it is immobilized on plastic (6). Electron micrographs of ␣-SNAP bound to SNARE complex and an antibody specific for the N terminus of ␣-SNAP show that ␣-SNAP binds SNARE in an antiparallel orientation. This positions its N terminus near the membrane and its C terminus away from the membrane, where it can interact with NSF (2, 7). Consistent with this, the ␣-SNAP C terminus has been shown to play a critical role in stimulating NSF ATPase activity and promoting SNARE complex disassembly (8).Despite limited overall sequence similarity among SNAREs on different...

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Section: Snare Complex Binding By ␣-Snapsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 79%

Section: Snare Complex Binding By ␣-Snapmentioning

confidence: 88%

mentioning

confidence: 99%

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Defining the SNARE Complex Binding Surface of α-SNAP

Marz

Lauer

Hanson

2003

Journal of Biological Chemistry

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“…p115 function is facilitated by interaction with two other Golgin family tethers, GM130 and giantin 35,39,51-53. Consistent with its central role in membrane organization, p115 is a target for apoptotic caspases 3 and 8 that leads to inactivation and fragmentation of the exocytic pathway and amplification of programmed cell death54,55 Numerous molecular and biochemical studies suggest that cytosolic p115 is anchored to membranes by the Ras-superfamily GTPase Rab1 and is involved in the assembly of SNARE complexes to promote bilayer fusion 35,37,43,47,48,56,57. The structural mechanism(s) by which p115 functions to promote tethering and SNARE assembly is unknown.…”

Section: Introductionmentioning

confidence: 98%

Structural and Functional Analysis of the Globular Head Domain of p115 Provides Insight into Membrane Tethering

Chen

Moyer

et al. 2009

Journal of Molecular Biology

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Molecular tethers play a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 Å resolution. The p115 structures show a novel α-solenoid architecture constructed of 12 armadillo-like, tether-repeat (TR), α-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in ER to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of ER to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes.

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