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Nef, an accessory protein of human and simian immunodeficiency viruses, is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, we developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. We found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. We also used a yeast three-hybrid system and glutathione S-transferase pull-down analyses to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4. Together, these results support a model in which HIV-1 Nef downregulates CD4 by promoting its accelerated endocytosis by a clathrin/AP2 pathway.

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination, and contains an intrinsic ubiquitin E3 ligase activity, all of which require an N-terminal A20 zinc finger and an immediately C-terminal helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5 Å resolution shows that Rabex-5:ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin binding domain, an inverted ubiquitin interaction motif (IUIM), that binds with ~29 μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ~22 μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc finger diaromatic patch mediates E3 ligase activity by directly recruiting a ubiquitin-loaded ubiquitin conjugating enzyme.The Rab GTPases are central regulators of vesicular trafficking and organelle identity in all eukaryotes 1,2 . The Rab family is the largest branch of the Ras superfamily, comprising more than 60 members in mammalian cells. Like other small GTPases, the localization and activity of the Rab proteins is regulated by GTPase activating proteins (GAPs), guanine nucleotide dissociation inhibitors (GDIs), and guanine nucleotide exchange factors (GEFs) 3,4 . Rab GEFs promote the binding of GTP to Rab proteins, which in turn converts them to their active signaling conformation, and stabilizes their binding to cellular membranes. The founding member of the Rab5 GEF family is the yeast vacuolar sorting protein Vps9 5 . Vps9 is the yeast ortholog of the human Rab5 GEF Rabex-5. All Rab5 GEFs have in common a catalytic unit comprising a helical bundle and a Vps9 homology domain 6 . Most Rab5 GEFs do not function alone, but rather as components of larger multiprotein complexes, as exemplified by the Rabaptin-5-Rabex-5 complex 7-10 .Covalent monoubiquitination of proteins is a major regulatory signal in protein trafficking 11 . In this process, the C-terminal carboxylate of a single molecule of the highly conserved 76-amino acid protein ubiquitin is covalently linked to a Lys residue in a substrate protein.correspondence should be addressed to James H. Hurley at hurley@helix.nih.gov.. 4 Y. C. T. and R. M. contributed equally.Coordinates. The crystallographic coordinates have been deposited in the protein data bank with accession code ____ (to be provided). NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2006 September 26. Published in final edited form as:Nat Struct Mol Biol. 2006 March ; 13(3): 264-271. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis reaction is carried out by a series of enzymes known as E1, E2, and E3 12-14 . Monoubiquitination of many transmembrane cargo proteins marks them for sorting into endosomal pathways 15-17 . Monoubiquitin moieties on these proteins are recognized by specific ubiquitin binding domains in proteins of the tr...

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