The cytosolic C-terminus of the glucose transporter GLUT4 contains an acidic cluster endosomal targeting motif distal to the dileucine signal

The insulin-responsive glucose transporter GLUT4 is targeted to a post-endo cytic compartment in adipocytes, from where it moves to the cell surface in response to insulin. Previous Studies have identified two cytosolic target ing motifs that regulate the intracellular sequestration of this protein: F QQI(5-8) in the N-terminus and LL489.490 (one-letter amino acid notation) i n the C-terminus, In the present study we show that a GLUT4 chimaera in whi ch the C-terminal 12 amino acids in GLUT4 have been replaced with the same region from human GLUT3 is constitutively targeted to the plasma membrane w hen expressed in 3T3-L1 adipocytes. To further dissect this domain it was d ivided into three regions, each of which was mutated en bloc to alanine res idues. Analysis of these constructs revealed that the targeting information is contained within the residues TELEYLGP(498-505). Using the transferrin- horseradish peroxidase endosomal ablation technique in 3T3-L1 adipocytes, w e show that mutants in which this C-terminal domain has been disrupted are more sensitive to chemical ablation than wild-type GLUT4. These data indica te that GLUT4 contains a targeting signal in its C-terminus, distal to the dileucine motif, that regulates its sorting into a post-endosomal compartme nt. Similar membrane-distal, acidic-cluster-based motifs are found in the c ytosolic tails of the insulin-responsive aminopeptidase IRAP (insulin-regul ated aminopeptidase) and the proprotein convertase PC6B, indicating that th is type of motif may play an important role in the endosomal sequestration of a number of different proteins.