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Novel putative targets of N ‐ethylmaleimide sensitive fusion protein (NSF) and α/β soluble NSF attachment proteins (SNAPs) include the Pak‐binding nucleotide exchange factor βPIX
Author(s) -
Martin Henry G.S.,
Henley Jeremy M.,
Meyer Guido
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20998
Subject(s) - gene isoform , fusion protein , microbiology and biotechnology , leucine zipper , plasma protein binding , biology , binding protein , chaperone (clinical) , biochemistry , chemistry , recombinant dna , peptide sequence , gene , medicine , pathology
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 (α, β and γ) are known. Here, we sought to identify novel targets of the NSF‐SNAP complex. A yeast two‐hybrid screen using the brain specific βSNAP 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 βSNAP. A yeast two‐hybrid screen using NSF as bait identified Rab11‐FIP3 and the Pak‐binding nucleotide exchange factor βPIX as putative binding partners. βPIX 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 βPIX mediates binding to NSF; however, this fragment of βPIX 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. J. Cell. Biochem. 99: 1203–1215, 2006. © 2006 Wiley‐Liss, Inc.