Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulininduced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c −/−
SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.
When microbes sense environmental changes, they often temporarily attenuate cell growth to adapt to the new situations, showing a lag phase. In this study, we report that the methylotrophic yeast, Pichia pastoris, induced autophagy during the lag phase after the cells were shifted from glucose to methanol medium. Through the autophagic process at least two proteins, aminopeptidase I precursor and cytosolic aldehyde dehydrogenase, were found to be transported into the vacuole, which was dependent on PpAtg11 and PpAtg17, respectively. Notably, PpAtg1 and PpAtg17 were required for early exit from the lag phase during the methanol adaptation. In accordance, phosphorylation states of elongation initiation factor 2α indicated reductions of intracellular amino-acid pools in the atg mutant strains. Together, these data demonstrate the importance of amino acid recycling by autophagy during a cell-remodeling process.
Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulininduced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c −/−
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