The insulin-regulatable glucose transporter (IRGT) is specifically expressed in muscle and fat cells and undergoes translocation from an intracellular compartment to the cell surface following acute insulin treatment. This study examined sorting differences between the IRGT and the homologous HepG2/erythrocyte/brain glucose transporter (HepG2 GT) when expressed together in insulin-responsive 3T3-L1 adipocytes. The ratio of the amount of transporter per unit protein in the plasma membrane fraction vs. the intracellular membrane fraction was 1:2 for the HepG2 GT and 1:30 for the IRGT. Insulin treatment increased the plasma membrane concentration of the IRGT by 10-fold and the HepG2 GT by 3.5-fold. This distribution was confirmed by confocal immunofluorescence microscopy. Differential sorting within intracellular organelles was evident by sucrose gradient analysis and immunoisolation of transporter vesicles and by double immunofluorescence labeling. We propose that differential sorting at an intracellular locus preferably withdraws the IRGT from a pathway which is in close communication with the plasma membrane, thus allowing the IRGT to regulate glucose entry into fat and muscle cells in a highly insulin-regulated fashion.

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.DOI: http://dx.doi.org/10.7554/eLife.00828.001

Ubiquitination functions as a sorting signal for lysosomal degradation of cell-surface proteins by facilitating their internalization from the plasma membrane and incorporation into lumenal vesicles of multivesicular bodies (MVBs). Ubiquitin may also mediate sorting of proteins from the trans-Golgi network (TGN) to the endosome, thereby preventing their appearance on the cell surface and hastening their degradation in the lysosome-vacuole. Substantiation of a direct ubiquitin-dependent TGN sorting pathway relies in part on identifying candidate machinery that may function as a ubiquitin-sorting 'receptor'at the TGN. Members of the GGA family of coat proteins localize to the TGN and promote the incorporation of proteins into clathrin-coated vesicles destined for transport to endosomes. We show that the GGA coat proteins bind directly to ubiquitin through their GAT domain and demonstrate that this interaction is required for the ubiquitin-dependent sorting of the Gap1 amino acid transporter from the TGN to endosomes. Thus, GGA proteins fulfill the role of ubiquitin sorting receptors at the TGN.

. Insulin stimulates glucose transport in adipocytes via the rapid redistribution of the GLUT! and GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. Insulin sensitivity is dependent on the proper intracellular trafficking of the glucose transporters in the basal state. The bulk of insulin-sensitive transport in adipocytes appears to be due to the translocation of GLUT4, which is more efficiently sequestered inside the cell and is present in much greater abundance than GLUTI.The cell type and isoform specificity of GLUT4 intracellular targeting were investigated by examining the subcellular distribution of GLUTI and GLUT4 in cell types that are refractory to the effect of insulin on glucose transport. Rat GLUT4 was expressed in 3T3-LI fibroblasts and HepG2 hepatoma cells by DNAmediated transfection . Transfected 3T3-Ll fibroblasts over-expressing human GLUTI exhibited increased glucose transport, and laser confocal immunofluorescent imaging of GLUTI in these cells indicated that the protein was concentrated in the plasma membrane. In contrast, 3T3-Ll fibroblasts expressing GLUT4 exhibited no increase in transport activity, and confocal imaging demonstrated that this protein was targeted almost exclusively to cytoplasmic compartments . 3T3-Ll fibroblasts expressing GLUT4 were unresponsive to r.UCOSE, an essential nutrient for many mammalian cells, is transported across the plasma membrane via facilitative carrier proteins . Five glucose transporter isoforms have been described thus far (for reviews, see Gould and Bell, 1990;Mueckler, 1990). These are named GLUTI through GLUT5 in order of their identification by means of cDNA cloning. The five transporter isoforms are distinguished by their discrete tissue distributions, kinetic properties, and regulation . In most cell types, such as endothelial cells, hepatocytes, and parenchymal cells of the brain, glucose transport is probably not rate limiting for glu-

In the late endocytic pathway, it has been proposed that endocytosed macromolecules are delivered to a proteolytic environment by 'kiss-and-run' events or direct fusion between late endosomes and lysosomes. To test whether the fusion hypothesis accounts for delivery to lysosomes in living cells, we have used confocal microscopy to examine content mixing between lysosomes loaded with rhodamine-dextran and endosomes subsequently loaded with Oregon-Green-dextran. Both kissing and explosive fusion events were recorded. Data from cell-free content-mixing assays have suggested that fusion is initiated by tethering, which leads to formation of a trans-SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) protein complex and then release of lumenal Ca2+, followed by membrane bilayer fusion. We have shown that the R-SNARE (arginine-containing SNARE) protein VAMP (vesicle-associated membrane protein) 7 is necessary for heterotypic fusion between late endosomes and lysosomes, whereas a different R-SNARE, VAMP 8 is required for homotypic fusion of late endosomes. After fusion of lysosomes with late endosomes, lysosomes are re-formed from the resultant hybrid organelles, a process requiring condensation of content and the removal/recycling of some membrane proteins.