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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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.[1]


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