HIV-1 Nef disrupts intracellular trafficking of major histocompatibility complex class I, CD4, CD8, and CD28 by distinct pathways that share common elements

The Nef protein is an important HIV virulence factor that promotes the degradation of host proteins to augment virus production and facilitate immune evasion. The best-characterized targets of Nef are major histocompatibility complex class I (MHC-I) and CD4, but Nef also has been reported to target several other proteins, including CD8β, CD28, CD80, CD86, and CD1d. To compare and contrast the effects of Nef on each protein, we constructed a panel of chimeric proteins in which the extracellular and transmembrane regions of the MHC-I allele HLA-A2 were fused to the cytoplasmic tails of CD4, CD28, CD8β, CD80, CD86, and CD1d. We found that Nef coprecipitated with and disrupted the expression of molecules with cytoplasmic tails from MHC-I HLA-A2, CD4, CD8β, and CD28, but Nef did not bind to or alter the expression of molecules with cytoplasmic tails from CD80, CD86, and CD1d. In addition, we used short interfering RNA (siRNA) knockdown and coprecipitation experiments to implicate AP-1 as a cellular cofactor for Nef in the downmodulation of both CD28 and CD8β. The interaction with AP-1 required for CD28 and CD8β differed from the AP-1 interaction required for MHC-I downmodulation in that it was mediated through the dileucine motif within Nef (LL(164,165)AA) and did not require the tyrosine binding pocket of the AP-1 μ subunit. In addition, we demonstrate a requirement for β-COP as a cellular cofactor for Nef that was necessary for the degradation of targeted molecules HLA-A2, CD4, and CD8. These studies provide important new information on the similarities and differences with which Nef affects intracellular trafficking and help focus future research on the best potential pharmaceutical targets.     

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.     

Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the mu but not the gamma/sigma subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical mu-binding motif. These data identify the mu subunit of AP-1 (mu1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with mu1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based mu-binding signal in the MHC-I CD by AP-1.     

ADP ribosylation factor 1 activity is required to recruit AP-1 to the major histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I trafficking in HIV-1-infected primary T cells

HIV-1-infected cells are partially resistant to anti-HIV cytotoxic T lymphocytes (CTLs) due to the effects of the HIV Nef protein on antigen presentation by major histocompatibility complex class I (MHC-I), and evidence has been accumulating that this function of Nef is important in vivo. HIV Nef disrupts the normal expression of MHC-I by stabilizing a protein-protein interaction between the clathrin adaptor protein AP-1 and the MHC-I cytoplasmic tail. There is also evidence that Nef activates a phosphatidylinositol 3 kinase (PI3K)-dependent GTPase, ADP ribosylation factor 6 (ARF-6), to stimulate MHC-I internalization. However, the relative importance of these two pathways is unclear. Here we report that a GTPase required for AP-1 activity (ARF-1) was needed for Nef to disrupt MHC-I surface levels, whereas no significant requirement for ARF-6 was observed in Nef-expressing T cell lines and in HIV-infected primary T cells. An ARF-1 inhibitor blocked the ability of Nef to recruit AP-1 to the MHC-I cytoplasmic tail, and a dominant active ARF-1 mutant stabilized the Nef-MHC-I-AP-1 complex. These data support a model in which Nef and ARF-1 stabilize an interaction between MHC-I and AP-1 to disrupt the presentation of HIV-1 epitopes to CTLs.     

Adaptor Protein 1 Promotes Cross-Presentation through the Same Tyrosine Signal in Major Histocompatibility Complex Class I as That Targeted by HIV-1

Certain antigen-presenting cells (APCs) process and present extracellular antigen with major histocompatibility complex class I (MHC-I) molecules to activate naive CD8⁺ T cells in a process termed cross-presentation. We used insights gained from HIV immune evasion strategies to demonstrate that the clathrin adaptor protein adaptor protein 1 (AP-1) is necessary for cross-presentation by MHC-I molecules containing a cytoplasmic tail tyrosine signal (murine MHC-I molecules, human MHC-I HLA-A and HLA-B allotypes). In contrast, AP-1 activity was not needed for cross-presentation by MHC-I molecules containing a human MHC-I HLA-C cytoplasmic tail, which does not contain a tyrosine signal. AP-1 activity was also dispensable for presentation of endogenous antigens by MHC-I via the classical pathway. In APCs, we show that HIV Nef disrupts cross-presentation by MHC-I containing the tyrosine signal but does not affect cross-presentation by MHC-I containing the HLA-C cytoplasmic tail. Thus, we provide evidence for two separable cross-presentation pathways, only one of which is targeted by HIV.     

Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.     

An MHC-I Cytoplasmic Domain/HIV-1 Nef Fusion Protein Binds Directly to the μ Subunit of the AP-1 Endosomal Coat Complex

Background

The down-regulation of the major histocompatibility complex class I (MHC-I) from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD) of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1). The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxφ, which mediates binding to the medium (μ) subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the μ subunit of AP-1 (μ1) as if it contained a Yxxφmotif.

Methods and Findings

Here, we show that a direct interaction between the MHC-I CD/Nef and μ1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of μ1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and μ1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on μ1 for Yxxφ motifs were required for a robust interaction.

Conclusions

These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the μ subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in μ1 for interaction with MHC-I CD/Nef.     

The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2

In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.     

A diacidic motif in human immunodeficiency virus type 1 Nef is a novel determinant of binding to AP-2

A key function of the Nef protein of immunodeficiency viruses is the downregulation of the T-cell and macrophage coreceptor, CD4, from the surfaces of infected cells. CD4 downregulation depends on a conserved (D/E)XXXL(L/I)-type dileucine motif in the C-terminal, flexible loop of Nef, which mediates binding to the clathrin adaptor complexes AP-1, AP-2, and AP-3. We now report the identification of a consensus (D/E)D motif within this loop as a second, conserved determinant of interaction of Nef with AP-2, though not with AP-1 and AP-3. Mutations in this diacidic motif abrogate both AP-2 binding and CD4 downregulation. We also show that a dileucine motif from tyrosinase, both in its native context and in the context of Nef, can bind to AP-2 independently of a diacidic motif. These results thus identify a novel type of AP-2 interaction determinant, support the notion that AP-2 is the key clathrin adaptor for the downregulation of CD4 by Nef, and reveal a previously unrecognized diversity among dileucine sorting signals.     

HIV-1 Nef-induced down-regulation of MHC class I requires AP-1 and clathrin but not PACS-1 and is impeded by AP-2

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment.     

Distinct trafficking pathways mediate Nef-induced and clathrin-dependent major histocompatibility complex class I down-regulation

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.     

AP-1 and AP-3 facilitate lysosomal targeting of Batten disease protein CLN3 via its dileucine motif

CLN3 is a transmembrane protein with a predominant localization in lysosomes in non-neuronal cells but is also found in endosomes and the synaptic region in neuronal cells. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinosis or Batten disease, which currently is the most common cause of childhood dementia. We have recently reported that the lysosomal targeting of CLN3 is facilitated by two targeting motifs: a dileucine-type motif in a cytoplasmic loop domain and an unusual motif in the carboxyl-terminal cytoplasmic tail comprising a methionine and a glycine separated by nine amino acids (Kyttala, A., Ihrke, G., Vesa, J., Schell, M. J., and Luzio, J. P. (2004) Mol. Biol. Cell 15, 1313-1323). In the present study, we investigated the pathways and mechanisms of CLN3 sorting using biochemical binding assays and immunofluorescence methods. The dileucine motif of CLN3 bound both AP-1 and AP-3 in vitro, and expression of mutated CLN3 in AP-1- or AP-3-deficient mouse fibroblasts showed that both adaptor complexes are required for sequential sorting of CLN3 via this motif. Our data indicate the involvement of complex sorting machinery in the trafficking of CLN3 and emphasize the diversity of parallel and sequential sorting pathways in the trafficking of membrane proteins.     

Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.     

The cytoplasmic tail of the cation-independent mannose 6-phosphate receptor contains four binding sites for AP-1

The trafficking of the cation-independent mannose 6-phosphate receptor between the trans-Golgi network and endosomes requires binding of sorting determinants in the cytoplasmic tail of the receptor to adaptor protein complex-1 (AP-1). Using a GST pull-down binding assay, four binding motifs were identified in the cytoplasmic tail: a tyrosine-based motif ((26)YSKV(29)), an internal dileucine-based motif ((39)ETEWLM(44)), and two casein kinase 2 sites ((84)DSEDE(88) and (154)DDSDED(159)). The YSKV motif mediated the strongest interaction with AP-1 and the two CK2 motifs bound AP-1 only when they were phosphorylated. The COOH-terminal dileucines were not required for interaction with AP-1.     

HIV-1 Nef targets MHC-I and CD4 for degradation via a final common beta-COP-dependent pathway in T cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7(+) vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, beta-COP. Moreover, we demonstrate that Nef contains two separable beta-COP binding sites. One site, an arginine (RXR) motif in the N-terminal alpha helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.     

Multiple C-terminal motifs of the 46-kDa mannose 6-phosphate receptor tail contribute to efficient binding of medium chains of AP-2 and AP-3

The interaction of adaptor protein (AP) complexes with signal structures in the cytoplasmic domains of membrane proteins is required for intracellular sorting. Tyrosine- or dileucine-based motifs have been reported to bind to medium chain subunits (mu) of AP-1, AP-2, or AP-3. In the present study, we have examined the interaction of the entire 67-amino acid cytoplasmic domain of the 46-kDa mannose 6-phosphate receptor (MPR46-CT) containing tyrosine- as well as dileucine-based motifs with mu2 and mu3A chains using the yeast two-hybrid system. Both mu2 and mu3A bind specifically to the MPR46-CT. In contrast, mu3A fails to bind to the cytoplasmic domain of the 300-kDa mannose 6-phosphate receptor. Mutational analysis of the MPR46-CT revealed that the tyrosine-based motif and distal sequences rich in acidic amino acid residues are sufficient for effective binding to mu2. However, the dileucine motif was found to be one part of a consecutive complex C-terminal structure comprising tyrosine and dileucine motifs as well as clusters of acidic residues necessary for efficient binding of mu3A. Alanine substitution of 2 or 4 acidic amino acid residues of this cluster reduces the binding to mu3A much more than to mu2. The data suggest that the MPR46 is capable of interacting with different AP complexes using multiple partially overlapping sorting signals, which might depend on posttranslational modifications or subcellular localization of the receptor.     

A noncanonical mu-1A-binding motif in the N terminus of HIV-1 Nef determines its ability to downregulate major histocompatibility complex class I in T lymphocytes

Downregulation of major histocompatibility complex class I (MHC-I) by HIV-1 Nef protein is indispensable for evasion of protective immunity by HIV-1. Though it has been suggested that the N-terminal region of Nef contributes to the function by associating with a mu-1A subunit of adaptor protein 1, the structural basis of the interaction between Nef and mu-1A remains elusive. We found that a tripartite hydrophobic motif (Trp13/Val16/Met20) in the N terminus of Nef was required for the MHC-I downregulation. Importantly, the motif functioned as a noncanonical mu-1A-binding motif for the interaction with the tyrosine motif-binding site of the mu-1A subunit. Our findings will help understanding of how HIV-1 evades the antiviral immune response by selectively redirecting the cellular protein trafficking system.     

Two sorting motifs, a ubiquitination motif and a tyrosine motif, are involved in HIV-1 and simian immunodeficiency virus Nef-mediated receptor endocytosis

HIV-1 and SIV Nef proteins downregulate cell surface CD4 and MHC class I (MHC-I) molecules of infected cells, which are necessary for efficient viral replication and pathogenicity. We previously reported that K144 in HIV-1 Nef is di-ubiquitinated, and K144R substitution impairs Nef-mediated CD4 downregulation. In this report, we extend the role of ubiquitination at this lysine residue from Nef-mediated CD4 downregulation to Nef-mediated MHC-I downregulation and from HIV Nef to SIV Nef. All HIV-1 Nef mutants that contain K144R substitution are inactive in MHC-I downregulation. Tested MHC-I alleles include HLA-ABC endogenously expressed and HLA-A2 exogenously expressed in Jurkat T cells. CD4 downregulation by SIV Nef involves K176 that aligns with K144 in HIV-1 Nef, as well as an N-terminal tyrosine motif Y28Y39 not present in HIV-1 Nef. Dual mutation at K176 and Y28Y39 completely impaired SIV Nef-mediated CD4 and MHC-I downregulation, whereas a single mutation at K176 or Y28Y39 did not. The involvement of tyrosine motif in SIV Nef-mediated CD4 and MHC-I downregulation prompted us to investigate a putative tyrosine motif (Y202Y/F203) in HIV-1 Nef that is conserved among HIV-1 species. Single mutation at the tyrosine motif Y202F203 in HIV-1 Nef (NA7) greatly impaired Nef-mediated CD4 downregulation, which is similar to what we observed previously with the single mutation at lysine K144. Thus, our study demonstrated that Nef-mediated receptor endocytosis involves the ubiquitination motif and tyrosine motif.     

HIV-1 Nef Targets MHC-I and CD4 for Degradation Via a Final Common β-COP–Dependent Pathway in T Cells

To facilitate viral infection and spread, HIV-1 Nef disrupts the surface expression of the viral receptor (CD4) and molecules capable of presenting HIV antigens to the immune system (MHC-I). To accomplish this, Nef binds to the cytoplasmic tails of both molecules and then, by mechanisms that are not well understood, disrupts the trafficking of each molecule in different ways. Specifically, Nef promotes CD4 internalization after it has been transported to the cell surface, whereas Nef uses the clathrin adaptor, AP-1, to disrupt normal transport of MHC-I from the TGN to the cell surface. Despite these differences in initial intracellular trafficking, we demonstrate that MHC-I and CD4 are ultimately found in the same Rab7⁺ vesicles and are both targeted for degradation via the activity of the Nef-interacting protein, β-COP. Moreover, we demonstrate that Nef contains two separable β-COP binding sites. One site, an arginine (RXR) motif in the N-terminal α helical domain of Nef, is necessary for maximal MHC-I degradation. The second site, composed of a di-acidic motif located in the C-terminal loop domain of Nef, is needed for efficient CD4 degradation. The requirement for redundant motifs with distinct roles supports a model in which Nef exists in multiple conformational states that allow access to different motifs, depending upon which cellular target is bound by Nef.     

AP-1 recruitment to VAMP4 is modulated by phosphorylation-dependent binding of PACS-1

The R-SNARE VAMP4, which contains a dileucine motif, binds to the AP-1 (adaptor protein-1) subunit μ1a, but not μ1b, or the GGAs (Golgi-associated gamma ear containing ARF binding proteins). Serine 20 and leucines 25,26 are essential for this binding. AP-1 association with VAMP4 is enhanced when serine 30, in an acidic cluster, is phosphorylated by casein kinase 2. This phosphorylation-dependent modulation of AP-1 binding is mediated by PACS-1 (phosphofurin acidic cluster sorting protein). Ablation of both the dileucine motif and serine 30 results in a dramatic mislocalization of VAMP4 in the regulated secretory pathway in AtT20 cells. A dominant-negative PACS-1, which binds acidic clusters but not AP-1, also causes mislocalization of VAMP4. Our data support a model whereby phosphorylation-dependent recruitment of PACS-1 enhances AP-1 association to cargo, and suggest that efficient retrieval depends on the formation of a complex between cargo, such as VAMP4, AP-1 and PACS-1.