Novel putative targets of N-ethylmaleimide sensitive fusion protein (NSF) and alpha/beta soluble NSF attachment proteins (SNAPs) include the Pak-binding nucleotide exchange factor betaPIX.
N-ethylmaleimide sensitive fusion protein (NSF) is a chaperone that plays a crucial role in the fusion of vesicles with target membranes. NSF mediates the ATP-consuming dissociation of a core protein complex that assembles during vesicle fusion and it thereby recharges the fusion machinery to perform multiple rounds of fusion. The binding of NSF to the core complex is mediated by co-chaperones named soluble NSF attachment proteins (SNAPs), for which three isoforms (alpha, beta and gamma) are known. Here, we sought to identify novel targets of the NSF-SNAP complex. A yeast two-hybrid screen using the brain specific betaSNAP isoform as bait revealed, as expected, NSF and several isoforms of the SNARE protein syntaxin as interactors. In addition, three isoforms of the reticulon protein family and two isoforms of BNIP3 interacted with betaSNAP. A yeast two-hybrid screen using NSF as bait identified Rab11-FIP3 and the Pak-binding nucleotide exchange factor betaPIX as putative binding partners. betaPIX interacts with recombinant NSF in co-sedimentation assays and the two proteins may be co-immunoprecipitated. A leucine zipper (LZ) motif within the C-terminus of betaPIX mediates binding to NSF; however, this fragment of betaPIX does not exhibit dominant negative effects in a cellular assay. In summary, our results support the evolving view that NSF has numerous targets in addition to conventional SNARE complexes.
Martin
Henry G S
HG
Medical Research Council Centre for Synaptic Plasticity, Department of Anatomy, University of Bristol, Bristol BS8 1TD, United Kingdom. henrym@interchange.ubc.ca
Henley
Jeremy M
JM
Meyer
Guido
G
D000818:Animals; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D049229:Dendritic Spines / Q000378:metabolism; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism*; D051381:Rats; D064067:Rho Guanine Nucleotide Exchange Factors; D012995:Solubility; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D015003:Yeasts
D018797:Cell Cycle Proteins; D020662:Guanine Nucleotide Exchange Factors; D021122:Protein Subunits; D064067:Rho Guanine Nucleotide Exchange Factors; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins