EpsinR: an ENTH domain-containing protein that interacts with AP-1 - PubMed (original) (raw)

EpsinR: an ENTH domain-containing protein that interacts with AP-1

Jennifer Hirst et al. Mol Biol Cell. 2003 Feb.

Erratum in

Abstract

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.

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Figures

Figure 1

Figure 1

Identification of new binding partners for the γ appendage domain. (a) GST alone or a GST-γ appendage construct was incubated either with or without cytosol from A431 cells. Samples were subjected to SDS PAGE, the gel was stained with Coomassie blue, and the indicated bands were analyzed by MALDI-TOF mass spectometry. The matches were as follows: band 1, KIAA1414 or p200; band 2, two proteins, KIAA0171 or epsinR and Snx9; and band 3, FLJ10767 or human ARF GAP1. (b) Pulldowns were performed on A431 cell cytosol using either GST alone, GST fused to the α appendage, GST fused to the γ appendage, or GST fused to the GGA1 appendage. Blots were probed with the indicated antibodies.

Figure 2

Figure 2

Binding partners for the γ appendage domain in rat liver clathrin-coated vesicles. (a) Electron micrograph of a conventional thin section of clathrin-coated vesicles purified from rat liver. Scale bar, 200 nm. (b) SDS PAGE showing the purified clathrin-coated vesicles next to an equal protein loading of crude membranes from an earlier stage in the preparation. Clathrin heavy and light chains (CHC and CLC) and subunits of the AP-1 and AP-2 adaptor complexes are indicated. (c) Crude membranes and purified clathrin-coated vesicles were analyzed by Western blotting, using antibodies against γ-adaptin, epsinR, Snx9, and ARF GAP1. γ-Adaptin, epsinR, and Snx9 are all enriched in the purified coated vesicles. (d) Blots of crude membranes and purified clathrin-coated vesicles were probed with the GST-γ appendage domain construct followed by anti-GST. Two labeled bands, p75 and p50, are strongly enriched in the purified coated vesicles. (e) GST pulldowns of coated vesicle extracts. By MALDI-TOF mass spectrometry, both p75 and p50 were found to match epsinR.

Figure 3

Figure 3

Binding to appendage domains analyzed by ligand blotting and pulldown with an epsinR domain. (a) Schematic diagrams of epsinR, Snx9, and GAP1, showing the positions of the various domains. (b) Snx9, the epsinR COOH-terminal domain (epsinR C), the epsinR NH2-terminal ENTH domain (epsinR N), and the GAP1 COOH-terminal domain (GAP1 C) were expressed as NH2-terminally His/Xpress-tagged constructs and analyzed by Western blotting. Blots were probed either with anti-Xpress or with the indicated GST constructs. The ability of the appendage domains to bind on blots indicates that they all recognize short linear motifs. (c) A construct consisting of GST fused to amino acids 324–428 of epsinR was used to pull down proteins from A431 cell cytosol, the gel was stained with Coomassie blue, and selected bands were analyzed by MALDI-TOF mass spectrometry. The indicated bands were found to contain clathrin heavy chain (CHC) and the AP-1 β1 and γ subunits.

Figure 4

Figure 4

Localization of epsinR. (a) Western blot of homogenized A431 cells, either untreated (total) or centrifuged at high speed (sup and pellet), probed with affinity-purified antiepsinR. (b and c) COS cells were double-labeled for epsinR (b) and the AP-1 γ subunit (c). The two patterns show very good colocalization. (d and e) COS cells transiently transfected with myc-tagged p56 were treated with nocodazole for 2 h and then double-labeled either for epsinR (d, red in f) and γ-adaptin (e, green in f) or for epsinR (g, red in i) and tagged p56 (h, green in i). To quantify the extent of overlap, six cells double-labeled for epsinR and γ-adaptin and six cells double-labeled for epsinR and tagged p56 were analyzed, and individual spots were scored. Out of 1607 spots analyzed from the cells double-labeled for epsinR and γ-adaptin, 9% were positive for epsinR only, 7% were positive for γ-adaptin only, and 84% were positive for both proteins. Out of 1192 spots analyzed from the cells double-labeled for epsinR and p56, 48% were positive for epsinR only, 16% were positive for p56 only, and 36% were positive for both proteins. Scale bar, 20 μm.

Figure 5

Figure 5

(a and b) COS cells were transfected with GFP-tagged full-length epsinR (a) and then double-labeled for the AP-1 γ subunit (b). (c) Link to a video (Figure 5c.mov) showing two cells expressing GFP-tagged full-length epsinR. Frames were acquired every 3.25 s, and the total acquisition time for the entire sequence was 139 s. (d) A series of frames from the video showing an enlarged region of one of the cells. The arrow indicates a structure that is moving away from the Golgi region. Although the structure appears to tubulate, this is probably because it was moving while the pictures were taken. Scale bar: a and b, 12 μm; c, 40 μm; d, 10 μm.

Figure 6

Figure 6

Localization of individual epsinR domains. The COOH-terminal γ appendage binding domain (a) and the NH2-terminal ENTH domain (b and c) were each expressed as GFP constructs and transfected into COS cells. The COOH-terminal domain is cytosolic; however, the ENTH domain is localized to the plasma membrane and to a perinuclear compartment that shows partial overlap with the AP-1 γ subunit (d). Scale bar, 20 μm.

Figure 7

Figure 7

EM localization of the epsinR constructs. Cells expressing either GFP-tagged ENTH domain (a and b) or GFP-tagged full-length epsinR (c) were processed for immunogold EM. (a) Much of the ENTH domain construct is associated with the plasma membrane. (b) The ENTH domain construct is also associated with tubulovesicular membranes near the Golgi stack (labeled G). (c) Full-length epsinR has a similar distribution; in addition, it is often found on membranes covered with a thick coat that is probably clathrin (arrowheads). Scale bars, 500 nm.

Figure 8

Figure 8

The distribution of epsinR does not depend upon AP-1. (a–d) Cells from a μ1A knockout mouse, either transfected with wild-type μ1A (a and c) or nontransfected (b and d), were labeled with antibodies against either γ-synergin (a and b) or epsinR (c and d). (e–h) COS cells were treated with siRNA directed against μ1A and then double-labeled for either γ-adaptin (e) and γ-synergin (f) or for γ-adaptin (g) and epsinR (h). In both cases, γ-synergin becomes cytosolic in the affected cells, whereas epsinR stays membrane associated. (i–l) COS cells were transiently transfected with GFP coupled to the γ appendage domain (i and k) and double-labeled for either γ-synergin (j) or epsinR (l). Expression of this construct causes γ-synergin to become cytosolic; however, it has little effect on epsinR (see asterisks). (m–p) Cells were treated with 100 μg/ml BFA for 2 min and then double-labeled for either AP-1 (m) and γ-synergin (n) or for AP-1 (o) and epsinR (p). All three proteins redistribute in response to BFA. Scale bar: a–d, 14 μm; e–h, 25 μm; i–l, 18 μm; m–p, 20 μm.

Figure 9

Figure 9

Binding of the epsinR ENTH domain to phosphoinositides spotted onto a nitrocellulose filter. A phosphoinositide array was probed with a GST-ENTH domain construct. PtdIns(4)P is most strongly labeled, followed by PtdIns(5)P and PtdIns(3,4)P2. There is little or no significant labeling of PtdIns(4,5)P2.

Figure 10

Figure 10

In vitro recruitment of the epsinR ENTH domain. NRK cells were permeabilized by freezing and thawing and then incubated with pig brain cytosol containing recombinant Xpress-tagged epsinR ENTH domain, either with or without GTPγS and/or neomycin. Recruitment of the ENTH domain construct only occurs in the presence of GTPγS, indicating that it is ARF dependent. It is slightly blocked by neomycin, but the effect is less severe than on AP-2. Scale bar, 20 μm.

Figure 11

Figure 11

EpsinR depletion by RNAi. (a–d) HeLa cells (a and b) or COS cells (c and d) were incubated with epsinR siRNA for 3 d and then double-labeled for immunofluorescence with anti-epsinR (a and c) and anti–γ-adaptin (b and d). In both cases >90% of the cells had greatly reduced epsinR labeling. Scale bar, 20 μm. e, Western blots of HeLa and COS cells treated with either a control siRNA or epsinR siRNA were probed with anti-epsinR. (f) HeLa and COS cells, treated with either control, epsinR, or μ1A siRNA, were pulse chased with 35S, and both cell-associated (C) and secreted (S) cathepsin D were immunoprecipitated. The positions of the precursor (P) and mature (M) forms of the enzyme are marked. In the HeLa cells the ratio of secreted cathepsin D precursor to total cathepsin D is 0.158 in the control, 0.161 in the epsinR knockdown, and 0.377 in the μ1A knockdown. In the COS cells the ratios are 0.191 in the control, 0.196 in the epsinR knockdown, and 0.504 in the μ1A knockdown.

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