Premium
Discrimination of GLUT4 vesicle trafficking from fusion using a temperature‐sensitive Munc18c mutant
Author(s) -
Thurmond Debbie C.,
Pessin Jeffrey E.
Publication year - 2000
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/19.14.3565
Subject(s) - glut4 , biology , mutant , vesicle , chromosomal translocation , vesicle fusion , insulin , microbiology and biotechnology , biochemistry , endocrinology , gene , membrane , synaptic vesicle
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°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°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°C whereas Munc18c/TS bound syntaxin 4 only at 23°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°C followed by temperature shift to 37°C resulted in an increased rate of GLUT4 translocation compared with cells stimulated at 37°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.