Association of N-ethylmaleimide sensitive fusion (NSF) protein and soluble NSF attachment proteins-alpha and -gamma with glucose transporter-4-containing vesicles in primary rat adipocytes.
To investigate the role of N-ethylmaleimide sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAP)-containing fusion complexes in glucose transporter-4 (GLUT4) membrane trafficking, the subcellular distributions of NSF, alpha-SNAP, and gamma-SNAP in primary rat adipocytes were determined. A large fraction of the NSF and SNAPs were associated with intracellular membranes, distributed between the low-density microsomes (LDM) and high-density microsomes. Very little of the NSF and SNAPs were associated with the plasma membrane fraction. This distribution did not change after insulin stimulation. Approximately 75% of the NSF and SNAPs in the LDM fraction were coimmunoprecipitated with 85% of the GLUT4 and 60% of the vesicle associated membrane proteins (VAMPs; synaptobrevins) VAMP-2 and cellubrevin in anti-GLUT4 immunoadsorptions. In contrast to NSF and the SNAPs, the beta-coatomer protein (beta-COP) found in the LDM fraction was excluded from GLUT4 vesicles. When LDM fractions were solubilized with Thesit (octaethylene glycol dodecyl ether) or Triton X-100, approximately 40% of the alpha-SNAP was colocalized with NSF on glycerol gradients in large (approximately 20S), ATP-sensitive complexes. VAMP-2 and cellubrevin are concentrated in the LDM fractions and in GLUT4 vesicles; both were excluded from these complexes. These data suggest that the steady state association of NSF and the SNAPs with GLUT4 vesicles and cell membranes is independent of the formation of fusion complexes.
Mastick
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Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105, USA. masticc@aa.wl.com
Falick
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D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D020756:Coatomer Protein; D004822:Epididymis; D051275:Glucose Transporter Type 4; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D007425:Intracellular Membranes / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009124:Muscle Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D020756:Coatomer Protein; D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D050683:R-SNARE Proteins; C495694:Slc2a4 protein, rat; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat