Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant

Debbie C. Thurmond, Jeffrey E. Pessin

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3565-3575
Number of pages11
JournalEMBO Journal
Volume19
Issue number14
StatePublished - Jul 17 2000
Externally publishedYes

Fingerprint

Fusion reactions
Temperature
Qa-SNARE Proteins
Insulin
Cell membranes
Lysine
Arginine
Substitution reactions
Demonstrations
Cell Membrane
Proteins

Keywords

  • Adipocytes
  • Glucose transporter
  • Insulin
  • Munc18c
  • Vesicle trafficking

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

Cite this

Discrimination of GLUT4 vesicle trafficking from fusion using a temperature-sensitive Munc18c mutant. / Thurmond, Debbie C.; Pessin, Jeffrey E.

In: EMBO Journal, Vol. 19, No. 14, 17.07.2000, p. 3565-3575.

Research output: Contribution to journalArticle

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