Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant.
To examine the temporal relationship between pre- and post-docking events, we generated a Munc18c temperature-sensitive mutant (Munc18c/TS) by substitution of arginine 240 with a lysine residue. At the permissive temperature (23 degrees C), overexpression of both the wild type (Munc18c/WT) and the R240K mutant inhibited insulin-stimulated GLUT4/IRAP vesicle translocation. However, at the non-permissive temperature (37 degrees C) only Munc18c/WT inhibited GLUT4/IRAP translocation whereas Munc18c/TS was without effect. Moreover, Munc18c/WT bound to syntaxin 4 at both 23 and 37 degrees C whereas Munc18c/TS bound syntaxin 4 only at 23 degrees C. This was due to a temperature-dependent conformational change in Munc18c/TS, as its ability to bind syntaxin 4 and effects on GLUT4 translocation were rapidly reversible while protein expression levels remained unchanged. Furthermore, insulin stimulation of Munc18c/TS-expressing cells at 23 degrees C followed by temperature shift to 37 degrees C resulted in an increased rate of GLUT4 translocation compared with cells stimulated at 37 degrees C. To date, this is the first demonstration that the rate-limiting step for insulin-stimulated GLUT4 translocation is the trafficking of GLUT4 vesicles and not their fusion with the plasma membrane.
Thurmond
D C
DC
Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242-1109, USA.
Pessin
J E
JE
D016475:3T3 Cells; D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D010122:Cystinyl Aminopeptidase; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000494:pharmacology; D008239:Lysine / Q000235:genetics; D008561:Membrane Fusion / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D013696:Temperature; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism; D033921:Vesicular Transport Proteins
D051275:Glucose Transporter Type 4; D007328:Insulin; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D017527:Receptor, IGF Type 2; C495693:Slc2a4 protein, mouse; C497943:Stxbp3 protein, mouse; D033921:Vesicular Transport Proteins; D001120:Arginine; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D008239:Lysine