Clathrin-mediated endocytosis in AP-2–depleted cells

Motley, Alison M.; Bright, Nicholas A.; Seaman, Matthew N.; Robinson, Margaret S.

doi:10.1083/jcb.200305145

Cited by 640 publications

(807 citation statements)

References 35 publications

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“…5), indicating that γ1 and γ2 are mostly expressed as part of an AP-1 complex. In fact, it has been previously demonstrated that in the absence of one of the subunits, the respective AP complex becomes unstable and disassembles, resulting in decreased levels of the other subunits Meyer et al, 2000;Motley et al, 2003;Peden et al, 2002). Moreover, as for γ1, interaction of Nef with γ2 absolutely requires co-expression of the σ1 subunit (Mattera et al, 2011;and our results, data not shown).…”

Section: Discussionsupporting

confidence: 63%

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

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Section: Discussionsupporting

confidence: 63%

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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“…Pulldown assays have also suggested that LL 814/815 and LL 855/856 bind AP-1 (Wyss et al, 2001), but no previous evidence has suggested that either of these motifs bind AP-2 or that the C terminal LL motif can mediate endocytosis of HIV Env. To examine the functional activities of these signals in more detail, we used siRNA against the 2 subunit of AP-2, which have been demonstrated to inhibit AP-2-mediated endocytosis (Fraile-Ramos et al, 2003;Motley et al, 2003). HeLa cells expressing CD4-Env constructs were treated with the siRNA, and the levels of 2 expression were analyzed 56 h after nucleofection by Western blot.…”

Section: Endocytosis Of Cd4/hiv Env Chimeras Is Dependent On the Clatmentioning

confidence: 99%

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

Byland

Vance

Hoxie

et al. 2007

MBoC

124

133

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During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxxØ motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV mac239 Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV HxB2 also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxxØ motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV mac239 Env GYxxØ motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions. INTRODUCTIONThe assembly of enveloped animal viruses requires that the viral and cellular components needed to make infectious particles are brought to the same site within the infected cell at the same time. For the primate lentiviruses (the human immunodeficiency viruses types 1 and 2 [HIV-1 and -2] and the simian immunodeficiency viruses [SIV]) the key viral structural proteins required to generate infectious particles are Gag, Gag-Pol, and Env. Although Gag alone can promote the formation of virus-like particles, Env and Gag-Pol are both essential for the formation of infectious virions. Although a considerable amount is known about these proteins, little is understood of the mechanisms through which they are targeted to the sites of assembly in infected cells.In many cell types, HIV assembles at the plasma membrane and, in the course of Gag assembly, the particles derive their membrane from the plasma membrane. For these particles to be infectious, Env must be transported to the cell surface, but the level to which it is incorporated into budding virions is unclear. Recent data have suggested that fewer than 10 Env protein complexes (trimers) are incorporated per virion (Chertova et al., 2002;Zhu et al., 2003), and early studies of HIV infected T-cells showed that much of the newly synthesized Env is transported to lysosomes and degraded (Willey et al., 1988). However, it has recently become evident that in macrophages the assembly of Envcontaining infectious HIV occurs on intracellular membranes, that have some characteristics in common with late endosomes (Raposo et al., 2002;Pelchen-Matthews et al., 2003). The assembly of virus in distinct...

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“…siRNAs against clathrin and 1A have been described (Hirst et al, 2003;Motley et al, 2003). HeLa cells were transfected using Oligofectamine (Invitrogen, Carlsbad, CA) as specified by the manufacturer.…”

Section: Rna Interferencementioning

confidence: 99%

EpsinR Is an Adaptor for the SNARE Protein Vti1b

Hirst

Miller

Taylor

et al. 2004

MBoC

Self Cite

124

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EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a ϳ50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR-and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

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Clathrin-mediated endocytosis in AP-2–depleted cells

Cited by 640 publications

References 35 publications

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

A Conserved Dileucine Motif Mediates Clathrin and AP-2–dependent Endocytosis of the HIV-1 Envelope Protein

EpsinR Is an Adaptor for the SNARE Protein Vti1b

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