Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles.
To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.
Thurmond
D C
DC
Department of Physiology, The University of Iowa, Iowa City, Iowa 52242, USA.
Kanzaki
M
M
Khan
A H
AH
Pessin
J E
JE
D017667:Adipocytes / Q000378:metabolism*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D010122:Cystinyl Aminopeptidase; D003594:Cytoplasmic Granules / Q000378:metabolism; D051275:Glucose Transporter Type 4; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D051938:Munc18 Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; C495693:Slc2a4 protein, mouse; C497943:Stxbp3 protein, mouse; D033921:Vesicular Transport Proteins; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase