Epidermal Growth Factor-Dependent Phosphorylation of the GGA3 Adaptor Protein Regulates Its Recruitment to Membranes

The Golgi-localized, Gamma-ear-containing, Arf-binding (GGA) proteins are monomeric clathrin adaptors that mediate the sorting of transmembrane cargo at the trans-Golgi network and endosomes. Here we report that one of these proteins, GGA3, becomes transiently phosphorylated upon activation of the epidermal growth factor (EGF) receptor. This phosphorylation takes place on a previously unrecognized site in the “hinge” segment of the protein, S368, and is strictly dependent on the constitutive phosphorylation of another site, S372. The EGF-induced phosphorylation of S368 does not require internalization of the EGF receptor or association of GGA3 with membranes. This phosphorylation can be blocked by inhibitors of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways that function downstream of the activated EGF receptor. Phosphorylation of GGA3 on S368 causes an increase in the hydrodynamic radius of the protein, indicating a transition to a more asymmetric shape. Mutation of S368 and S372 to a phosphomimic aspartate residue decreases the association of GGA3 with membranes. These observations indicate that EGF signaling elicits phosphorylation events that regulate the association of GGA3 with organellar membranes

Divalent interaction of the GGAs with the Rabaptin-5–Rabex-5 complex

Cargo transfer from trans-Golgi network (TGN)-derived transport carriers to endosomes involves a still undefined set of tethering/fusion events. Here we analyze a molecular interaction that may play a role in this process. We demonstrate that the GGAs, a family of Arf-dependent clathrin adaptors involved in selection of TGN cargo, interact with the Rabaptin-5–Rabex-5 complex, a Rab4/Rab5 effector regulating endosome fusion. These interactions are bipartite: GGA-GAE domains recognize an FGPLV sequence (residues 439–443) in a predicted random coil of Rabaptin-5 (a sequence also recognized by the γ1- and γ2-adaptin ears), while GGA-GAT domains bind to the C-terminal coiled-coils of Rabaptin-5. The GGA–Rabaptin-5 interaction decreases binding of clathrin to the GGA-hinge domain, and expression of green fluorescent protein (GFP)–Rabaptin-5 shifts the localization of endogenous GGA1 and associated cargo to enlarged early endosomes. These observations thus identify a binding sequence for GAE/γ-adaptin ear domains and reveal a functional link between proteins regulating TGN cargo export and endosomal tethering/fusion events

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

<div><p>Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons.</p></div

A Basic Patch on α-Adaptin Is Required for Binding of Human Immunodeficiency Virus Type 1 Nef and Cooperative Assembly of a CD4-Nef-AP-2 Complex▿ †

A critical function of the human immunodeficiency virus type 1 Nef protein is the downregulation of CD4 from the surfaces of infected cells. Nef is believed to act by linking the cytosolic tail of CD4 to the endocytic machinery, thereby increasing the rate of CD4 internalization. In support of this model, weak binary interactions between CD4, Nef, and the endocytic adaptor complex, AP-2, have been reported. In particular, dileucine and diacidic motifs in the C-terminal flexible loop of Nef have been shown to mediate binding to a combination of the α and σ2 subunits of AP-2. Here, we report the identification of a potential binding site for the Nef diacidic motif on α-adaptin. This site comprises two basic residues, lysine-297 and arginine-340, on the α-adaptin trunk domain. The mutation of these residues specifically inhibits the ability of Nef to bind AP-2 and downregulate CD4. We also present evidence that the diacidic motif on Nef and the basic patch on α-adaptin are both required for the cooperative assembly of a CD4-Nef-AP-2 complex. This cooperativity explains how Nef is able to efficiently downregulate CD4 despite weak binary interactions between components of the tripartite complex

Live-cell imaging shows that NiV-G increases axonal transport of NiV-F.

<p>(A–C) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s006" target="_blank">Video S1</a> (thin upper images) and kymographs (bottom images) of the analysis of particles moving along 100 µm of axons in DIV10 rat hippocampal neurons co-transfected on DIV5 with plasmids encoding either NiV-F-GFP (NiV-F) and mCh-Tub (Tub) (panel A), NiV-G-mCh (NiV-G) and GFP (panel B) or NiV-F-GFP and NiV-G-mCh (panel C). Images in (A) and (B) are shown in grayscale. In panel (C), NiV-F-GFP and NiV-G-mCh fluorescence are shown individually in grayscale and as green and red, respectively, in merged images (yellow indicates co-localization). Tracings with negative and positive slopes in kymographs represent anterograde and retrograde movement of particles, respectively; vertical lines represent particles that are stationary during the 60 s recording. (D) Quantification of NiV-F-GFP and NiV-G-mCh axonal transport in neurons expressing these proteins individually or in combination. Data shown represent the number of anterograde (Ant), stationary (Stat) and retrograde (Ret) particles per 100 µm of axon length during the 60 s recording. NiV-F and NiV-G (green and red bars, respectively) show the number of axonal particles containing these two proteins when expressed individually. “NiV-F (+NiV-G)” (light green bars) is the number of NiV-F-GFP particles in neurons co-expressing NiV-G-mCh; “NiV-G (+NiV-F)” (salmon bars) represents the number of NiV-G-mCh particles in neurons co-expressing NiV-F-GFP. Values are the means±SEM of 20–22 independent measurements for each condition and represent the total number of particles (n_p) indicated under the graph. Statistical significance was calculated by one-way ANOVA followed by Dunnett's test. (*) <i>P</i><0.01 when compared to NiV-F-GFP-containing particles in cells expressing only this protein. (E) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s007" target="_blank">Video S2</a> showing dendritic particles from neurons co-expressing either NiV-F-GFP and mCh-Tub, NiV-G-mCh and GFP, or NiV-F-GFP and NiV-G-mCh (upper panels in grayscale; bottom panel with merged NiV-F-GFP and NiV-G-mCh images in green and red, respectively). The lower color image is a 4× magnification of the boxed area in the above image; color arrows point to particles containing either NiV-F-GFP or NiV-G-mCh (green and red, respectively) or both proteins (yellow). Scale bar: 10 µm. (F) Co-localization of NiV-F-GFP and NiV-G-mCh in axonal and dendritic particles of co-transfected neurons. NiV-F-GFP co-localization in co-transfected neurons (NiV-F (+NiV-G), light green bars) was the percentage of NiV-F-GFP-containing particles that also contained NiV-G-mCh. Similarly, NiV-G-mCh co-localization (NiV-G (+NiV-F), salmon bars) was the percentage of NiV-G-mCh-containing particles that also displayed NiV-F-GFP. Values are the means±SEM of 22 and 33 measurements of axonal and dendritic particles, respectively, and represent the total number of axonal and dendritic particles (n_pA and n_pD) containing NiV-F-GFP and NiV-G-mCherry indicated under the graph.</p

Somatodendritic sorting of NiV-F mediated by its cytosolic tail.

<p>(A) Schematic representation of NiV-F and NiV-G indicating the NIV-F fusion peptide (FP), transmembrane (M) and cytosolic (C) domains, and amino-acid sequences of the cytosolic domains. The F₂-F₁ active form of NiV-F generated by cathepsin L- or B-mediated cleavage of the F₀ inactive precursor is stabilized by a disulfide (S-S) bridge <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Pager1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich2" target="_blank">[30]</a>. Utilized N-linked glycosylation sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Moll1" target="_blank">[26]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Biering1" target="_blank">[28]</a> are indicated by branched lines. The YXXØ motif (YSRL, residues 525–528) and a YY pair (residues 542 and 543) in the NiV-F tail are highlighted in red. (B–E) Rat hippocampal neurons were co-transfected on DIV4 with plasmids encoding NiV-F-GFP, NiV-G-HA, Tac or Tac-NiV-F, and mCherry-tubulin (mCh-Tub, marker of both dendrites and axons), fixed on DIV10, and immunostained with rabbit anti-MAP2 and goat anti-ankyrin-G (ANK-G) (to identify dendrites and the AIS, respectively) and with mouse anti-HA or mouse anti-Tac antibodies (to visualize NiV-G-HA or Tac-based constructs). Cells were imaged by confocal microscopy. Grayscale images correspond to NiV-F-GFP fluorescence (B), anti-HA (C) or anti-Tac (D, E) staining. Merged color pictures at the bottom of all panels display mCh-Tub fluorescence (red) and anti-MAP2 (green) staining (axons appear red due to mCh-Tub labeling, while dendrites are yellow due to co-labeling by mCh-Tub and MAP2). Insets show anti-ANK-G labeling (AIS shown in cyan). The AIS and axons are marked by cyan and red arrowheads, respectively. Scale bar: 20 µm. Quantitative analysis of NiV-F and NiV-G polarized sorting was performed through calculation of the dendrite/axon (D/A) polarity index (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-t001" target="_blank">Table 1</a>).</p

Quantification of NiV-F and NiV-G sorting into somatodendritic and axonal domains of rat hippocampal neurons.

<p>Dendrite to axon (D/A) polarity indexes were calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#s4" target="_blank">Materials and Methods</a>. Values are expressed as mean <b>±</b> SD (<i>n</i>) (<i>n</i>; number of cells analyzed).</p><p>The notation NiV-F-GFP (+NiV-G-HA) refers to the NiV-F-GFP polarity index in cells co-expressing NiV-G-HA. The same applies to the NiV-G-HA (+NiV-F-GFP) notation.</p><p>Statistical significance for all groups including NiV-F-GFP and NiV-G-HA constructs was calculated by one-way ANOVA followed by Dunnett's test.</p><p>(*)<i>P</i><0.01 when compared to NiV-F-GFP. Significance between group pairs including Tac or NiV-F-GFP Δ104–109 constructs was calculated by Student's <i>t</i>-test.</p>(§)<p><i>P</i><0.01 when compared to Tac;</p>(‡)<p><i>P</i><0.01 when compared to NiV-F-GFP Δ104–109.</p

Interaction of the NiV-F cytosolic tail with AP μ subunits.

<p>(A) Scheme of heterotetrameric adaptor protein (AP) complexes depicting the four subunits in each complex along with subunit isoforms. Combinatorial assembly of subunits can originate multiple forms of AP-1, AP-2 and AP-3 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Mattera2" target="_blank">[56]</a>. (B) Y2H analysis showing interaction of the NiV-F cytosolic tail, but not the NiV-G cytosolic tail, with μ1A, μ1B, μ2, μ3A and μ4. Growth of yeast co-transformants on −His plates is indicative of interactions between tail constructs subcloned in a Gal4 binding domain (BD) vector and μ subunits subcloned in a Gal4 activation domain (AD) vector; growth on +His plates is a control for growth/loading of co-transformants. The TGN38 cytosolic tail was used as a positive control for interaction with various μ subunits. Co-transformations of cytosolic tail constructs with SV40 T-Ag and of μ subunits with p53 were used as negative controls. Co-transformation of p53 and SV40 T-Ag constructs provided an additional positive control for interactions. Images are composites of panels from the same experiments and are representative of three independent experiments. (C) Y2H analysis showing that alanine substitution of Y525 or L528 in the YXXØ-based signal or combined substitution of Y542 and Y543 inhibits the interaction of the NiV-F cytosolic tail with μ subunits. Experiments were performed as in panel B.</p