Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes

Abstract

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 epithelial polarity program: machineries involved and their hijacking by cancer

“…Basolateral sorting is guided by basolateral sorting signals (Keller and Simons, 1997;Rodriguez-Boulan et al, 2005), and they include: short tyrosine motifs such as Yxxf or NPXY (Brewer and Roth, 1991;Le Bivic et al, 1991;Matter et al, 1992), dileucine (Hunziker and Fumey, 1994) or monoleucine motifs with nearby acidic patches (Deora et al, 2004), PxxP, found in EGFR and pleomorphic motifs that do not yet fall into established classes, found in NCAM (Le Gall et al, 1994), transferrin receptor (Odorizzi and Trowbridge, 1997) and polymeric IgA receptor (Aroeti and Mostov, 1994) ( Figure 2). Yeast 2 and 3 hybrid assays (Ohno et al, 1995;Janvier et al, 2003) demonstrated that Yxxf motifs interact with the m subunit of AP adaptors (AP1, AP2, AP3, AP4), whereas dileucine motifs of the type [DE]XXXL [LI] bind to g and s1 subunits of AP1 and to d and s3 subunits of AP3 (Kirchhausen, 2000;Bonifacino and Traub, 2003). Crystallographic studies have elucidated details of some of these interactions, for example, of the AP m subunits with tyrosine motifs and of GGAs (Golgi-localized, g-ear containing, Arf-binding protein) with dileucine motifs in Man6PR (Bonifacino, 2004;Owen et al, 2004).…”

Antibody to AP1B Adaptor Blocks Biosynthetic and Recycling Routes of Basolateral Proteins at Recycling Endosomes

“…Trafficking in both biosynthetic and recycling routes is controlled by apical sorting signals (e.g., N-and O-glycans, lipid anchors, and protein domains with affinity for lipid rafts), by microtubule motor determinants (Rodriguez-Boulan and Gonzalez, 1999;Schuck and Simons, 2004;Rodriguez-Boulan et al, 2005) and by basolateral sorting signals (e.g., tyrosine, dileucine, and monoleucine motifs, some similar to endocytic determinants, and noncanonic motifs not yet matching any consensus sequence; Rodriguez-Boulan et al, 2005). Tyrosine-based signals are recognized by a family of organelle-specific tetrameric AP (adaptor protein) adaptors via their subunit, whereas dileucine motifs may be recognized via large (␥ and ␦) and small ( 1, 3) subunits of AP adaptors acting together (Bonifacino and Lippincott-Schwartz, 2003;Janvier et al, 2003).…”

“…Trafficking in both biosynthetic and recycling routes is controlled by apical sorting signals (e.g., N-and O-glycans, lipid anchors, and protein domains with affinity for lipid rafts), by microtubule motor determinants (Rodriguez-Boulan and Gonzalez, 1999;Schuck and Simons, 2004;Rodriguez-Boulan et al, 2005) and by basolateral sorting signals (e.g., tyrosine, dileucine, and monoleucine motifs, some similar to endocytic determinants, and noncanonic motifs not yet matching any consensus sequence; Rodriguez-Boulan et al, 2005). Tyrosine-based signals are recognized by a family of organelle-specific tetrameric AP (adaptor protein) adaptors via their subunit, whereas dileucine motifs may be recognized via large (␥ and ␦) and small ( 1, 3) subunits of AP adaptors acting together (Bonifacino and Lippincott-Schwartz, 2003;Janvier et al, 2003).The early paradigm of two separate sorting sites for proteins in biosynthetic or recycling pathways has, however, progressively shifted over the past decade to a different paradigm in which both TGN and RE share a sorting role in the biosynthetic route. This new paradigm emerged from the observations by several laboratories showing that newly synthesized PM proteins leaving the Golgi apparatus may traverse the endosomal compartment before arrival at the cell surface (Futter et al, 1995;Leitinger et al, 1995;Brachet et al, 1999;Orzech et al, 2000;Ang et al, 2003;Lock and Stow, 2005).…”

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

“…To examine direct interactions between Naked2 and adaptor proteins, we used Naked2 residues 1-173 and residues 1-265 (data not shown) in yeast two-hybrid analysis with components of the adaptor protein machinery generously provided by J. Bonifacino (Janvier et al, 2003). We observed no direct interactions between Naked2 and components of AP-1, AP-2, AP-3, and AP-4 under adenine minus (data not shown) or less stringent histidine minus growth conditions ( Figure 4E).…”

“…The adaptor subunits constructed in AD vectors were generously provided by Juan Bonifacino (National Institutes of Health) (Janvier et al, 2003). Naked2 N-terminal fragments were constructed in DNA-BD vector pGBKT7.…”