title: Botulinum E toxin light chain does not cleave SNAP-23 and only partially impairs insulin stimulation of GLUT4 translocation in 3T3-L1 cells.
abstract: The stimulation of glucose uptake into fat and muscle by insulin results predominantly from the translocation of the glucose transporter, GLUT4, from an intracellular vesicle pool to the cell surface. Homologues of several key proteins known to be involved in the process of synaptic vesicle fusion have been identified on GLUT4 vesicles, including VAMP2 and cellubrevin. Syntaxin 4, SNAP-23 and/or SNAP-25 are also implicated in this process. Bacterial toxins that specifically cleave these proteins have been utilised to assess their involvement in cell function. We aimed to distinguish which of the SNAP isoforms are specifically involved in GLUT4 translocation. Here we show that both human (h) and mouse (m) SNAP-23, unlike SNAP-25, are not substrates for Botulinum E toxin light chain (BoNT/E). Furthermore, we demonstrate that microinjection of differentiated 3T3-L1 cells with BoNT/E inhibited insulin stimulation of GLUT4 translocation only slightly, 27%, whereas tetanus toxin light chain, that cleaves VAMP2, inhibited insulin stimulation of GLUT4 translocation by 80%. These studies therefore do not support a major role for SNAP-25 in insulin stimulation of GLUT4 translocation and place SNAP-23 as a prime candidate for a role in this process.
authors.affiliation: Division of Molecular Science, CSIRO, 343 Royal Parade, Parkville, Victoria, 3052, Australia.
authors.forename: S L
authors.initials: SL
authors.lastname: Macaulay
authors.forename: S
authors.initials: S
authors.lastname: Rea
authors.forename: K H
authors.initials: KH
authors.lastname: Gough
authors.forename: C W
authors.initials: CW
authors.lastname: Ward
authors.forename: D E
authors.initials: DE
authors.lastname: James
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D001905:Botulinum Toxins / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000494:pharmacology*; D007329:Insulin Antagonists / Q000494:pharmacology*; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D007329:Insulin Antagonists; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011994:Recombinant Proteins; C495692:SLC2A4 protein, human; C100493:SNAP23 protein, human; C495693:Slc2a4 protein, mouse; C107017:Snap23 protein, mouse; D001905:Botulinum Toxins