Leucine-based motifs mediate the sorting of membrane proteins at such cellular sites as the trans-Golgi network, endosomes, and plasma membrane. A Leu paired with a second Leu, Ile, or Met, while itself lacking the ability to mediate transport, is the key structural feature in these motifs. Here we have studied the structural differences between the leucine-based motifs contained in the COOH tails of LIMPII and GLUT4, two membrane proteins that are transported through the secretory pathway and are targeted to lysosomes (1-3) and to a perinuclear compartment adjacent to the Golgi complex (4), respectively. LIMPII and GLUT4 display negatively (Asp 470 /Glu 471 ) and positively (Arg 484 /Arg 485 ) charged residues, respectively, at positions ؊4 and ؊5 upstream from the critical Leu residue. The change in the charge sign of residues ؊4 and ؊5 results in missorting of LIMPII and GLUT4. We note that the acidic Glu residue at position ؊4 is critical for efficient intracellular sorting of LIMPII to lysosomes, but is dispensable for its surface internalization by endocytosis. Efficient intracellular sorting and endocytosis of GLUT4 require an Arg pair between positions ؊4 and ؊7. These results are consistent with the existence of distinct leucine-based motifs and provide evidence of their different readings at different cellular sites.Leucine-based motifs are implicated in membrane protein sorting by clathrin devices at the TGN, 1 cell surface, and endosomes (Refs. 1-3 and 5-7; for reviews, see Refs. 8 and 9).Their structural determinants are, however, poorly characterized: a Leu paired with a second Leu, Ile, or Met is the only constant structural feature in these motifs, but the pair by itself lacks the ability to mediate transport (2, 8). Acidic residues located at positions Ϫ4 and Ϫ5 upstream from the Leu/ Leu(Ile) pair have been demonstrated to be required for efficient internalization of invariant chain (Ii) and CD4 chimeras in mammalian cells (see Table I) (10, 11) and for transport of the t-SNARE Vamp3 to yeast vacuoles (12). In addition, phosphorylation of a Ser residue at position 4 or 5 upstream is critical for the surface internalization of some membrane proteins (13-17). On the other hand, some of the Leu-based motifs lack upstream acidic residues in intracellular protein sorting and endocytosis, and this also strongly suggests the existence of a family of leucine-based motifs with distinct structural determinants and activities. The possibility of distinct specificity determinants that modulate the recognition of leucinebased motifs is also evident when the ability of specific clathrin adaptors to discriminate between leucine motifs is studied (11, 18 -22).Here we have studied the structural determinants involved in the Leu-mediated intracellular sorting and surface internalization of LIMPII and GLUT4, two membrane proteins with distinct cellular distributions. The results of our experiments indicate the existence of different Leu-based motifs endowed with distinct structural determinants. Furthermore, they...

Vaccinia virus (VV), the prototype member of the Poxviridae, a family of large DNA viruses, carries out DNA replication in specialized cytoplasmic sites that are enclosed by the rough endoplasmic reticulum (ER). We show that the VV gene product of A40R is quantitatively modified by SUMO-1, which is required for its localization to the ER-enclosed replication sites. Expression of A40R lacking SUMO-1 induced the formation of rod-shaped cytoplasmic aggregates. The latter likely consisted of polymers of nonsumoylated protein, because unmodified A40R interacted with itself, but not with the SUMO-1-conjugated protein. Using a bacterial sumoylation system, we furthermore show that unmodified A40R is mostly insoluble, whereas the modified form is completely soluble. By electron microscopy, the A40R rods seen in cells were associated with the cytosolic side of the ER and induced the apposition of several ER cisternae. A40R is the first example of a poxvirus protein to acquire SUMO-1. Its quantitative SUMO-1 modification is required for its proper localization to the viral "mini-nuclei" and prevents its self-association. The ability of the nonsumoylated A40R to bring ER membranes close together could suggest a role in the fusion of ER cisternae when these coalesce to enclose the VV replication sites.

The insulin-sensitive glucose transporter GLUT4 is translocated to the plasma membrane in response to insulin and recycled back to the intracellular store(s) after removal of the hormone. We have used clonal 3T3-L1 fibroblasts and adipocyte-like cells stably expressing wild-type GLUT4 to characterize (a) the intracellular compartment where the bulk of GLUT4 is intracellularly stored and (b) the mechanisms involved in the recycling of endocytosed GLUT4 to the store compartment. Surface internalized GLUT4 is targeted to a large, flat, fenestrated saccular structure resistant to brefeldin A that localized to the vicinity of the Golgi complex is sealed to endocytosed transferrin (GLUT4 storage compartment). Recycling of endocytosed GLUT4 was studied by comparing the cellular distributions of antibody/biotin tagged GLUT4 and GLUT4(Ser Plasma euglycemia is maintained by the effect of insulin on muscle and to a lesser extent on adipose tissue. In these tissues insulin stimulates glucose transport, the rate-limiting event in glucose disposal (1-3). The function of GLUT4, the only glucose transporter sensitive to insulin, is essential to maintain the insulin-regulated plasma euglycemia, which is controlled through the regulation of the GLUT4 trafficking. Insulin stimulates glucose transport by promoting the translocation of GLUT4 (4, 5) from the intracellular tubulovesicular structures where it is stored (6 -8) to the plasma membrane (5, 7, 9, 10). After withdrawal of the hormone, GLUT4 is removed from the plasma membrane and recycled to the intracellular stores, and the steady-state previous to insulin stimulation is re-established (11,12).The molecular mechanisms involved in GLUT4 trafficking are poorly understood. The 12 transmembrane domain protein displays, in its amino and carboxy-cytoplasmic tails, Phe 5 -Gln 6 -Gln 7 -Ile 8 -based (13) and Leu 489 -Leu 490 -based motifs (14) that when inactivated provoke its cellular redistribution. Both motifs have been involved in GLUT4 endocytosis, intracellular retention, and targeting in transfected 3T3-L1fibroblasts, COS-7 and Chinese hamster ovary cells (15-20), myoblasts (21), and adipocytes (18,(22)(23)(24). Whether the dileucine motif mediates the trafficking of GLUT4 in adipocytes is, however, the subject of much debate (18,22,23). In addition, the mechanisms that mediate the intracellular retention and recycling of GLUT4 to the intracellular store compartments remain essentially unknown.Here we have investigated the motifs involved in the recycling of endocytosed GLUT4 to the GSC 1 and study the organization and localization of this. For this purpose, we have stably expressed HA epitope-tagged wild-type GLUT4 and a GLUT4(Ser 5 ), a mutant with the Phe 5 -Gln 6 -Gln 7 -Ile 8 motif ablated, and studied the intracellular distribution of endocytosed molecules tagged with anti-HA antibodies and the GLUT4-specific reagent Bio-LC-ATB-BMPA. The results of these studies show that the Phe 5 -Gln 6 -Gln 7 -Ile 8 motif is critical for access of endocytosed GLUT4 to the GSC where ...