The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3.

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin-and to a lesser extent the other AP complexes-are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.Trafficking of membrane proteins is governed by a regulated machinery that involves the vesicular transport of proteins throughout different intracellular compartments. One major regulatory mechanism is related to the function of the adaptor protein (AP) 1 complexes that assemble on donor membranes of the endocytic pathway to form transport vesicles (for review, see Ref. 1). The sorting of transmembrane proteins into these vesicles requires the recognition by the AP complexes of specific tyrosine-or leucine-based motifs contained within the cytoplasmic domains of cargo proteins (2). Four different types of heterotetrameric AP complexes (AP-1-AP-4) have been identified (3). AP-2 is specifically involved in the formation of clathrincoated vesicles at the plasma membrane, whereas AP-1 and AP-3 mediate the formation of clathrin-coated vesicles at the levels of the trans-Golgi network (TGN) and endosomes. The function of AP-4 is less well documented, but it regulates formation of non-clathrin-coated vesicles at the TGN. The association of the AP-1, AP-3, and AP-4 complexes with TGN and endosomal membranes is regulated by ADP-ribosylation factor 1 (ARF1). The Nef protein of HIV-1 is a 27-kDa protein that associates with the cell membranes through N-terminal myristoylation and is abundantly produced shortly after virus infection (for review, see Refs. 4 and 5). Nef is an essential factor in vivo for efficient viral replication and pathogenesis. In vitro, Nef also facilitates virus replication and enhances the infectivity of virions. Although the positive influence of Nef on viral replication and infectivity may be multifactorial, genetic evidence suggests a relationship between these virological effects and the ability of Nef to modulate the cell surface expression of multiple membrane-associated proteins. In additio...