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Clathrin binding by the adaptor Ent5 promotes late stages of clathrin coat maturation.

Hung CW, Duncan MC - Mol. Biol. Cell (2016)

Bottom Line: We find that the direct binding of Ent5 with clathrin is required for its role in coat behavior and cargo traffic.Surprisingly, the interaction of Ent5 with other adaptors is dispensable for coat behavior but not cargo traffic.These findings support a model in which Ent5 clathrin binding performs a mechanistic role in coat maturation, whereas Ent5 adaptor binding promotes cargo incorporation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

No MeSH data available.


Related in: MedlinePlus

Clathrin is required for maximal interaction of Ent5 with Gga2 and localization of Ent5 to membranes. (A) A representative immunoprecipitation reaction. GFP-tagged Ent5 was immunoprecipitated from cell lysates, and the immunoprecipitates were probed with antibodies to Ent5, Gga2, and clathrin to monitor the effect of Ent5 mutations on the Ent5–Gga2 and Ent5–clathrin interactions. Apparent size shift in mutant proteins may be due to differences in surfactant binding, as previously observed for other mutations that alter charge (Shi et al., 2008; Rath et al., 2009; De Zutter et al., 2013). Quantification of the effects of Ent5 mutations on (B) Ent5–Gga2 interaction or (C) Ent5–Chc1 interaction (n = 3). Error bars indicate SD; p values determined with Student’s t test. (D) Chc1-box mutation blocks interaction of Ent5 with clathrin. Clc1 was immunoprecipitated from lysates of cells lacking CHC1 transformed with plasmids that contain wild-type CHC1 or chc1Δbox. (E) Live-cell imaging of CHC1-deleted cells transformed with plasmids that contain wild-type CHC1 or chc1Δbox mutant. Scale bar, 5 μm.
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Figure 4: Clathrin is required for maximal interaction of Ent5 with Gga2 and localization of Ent5 to membranes. (A) A representative immunoprecipitation reaction. GFP-tagged Ent5 was immunoprecipitated from cell lysates, and the immunoprecipitates were probed with antibodies to Ent5, Gga2, and clathrin to monitor the effect of Ent5 mutations on the Ent5–Gga2 and Ent5–clathrin interactions. Apparent size shift in mutant proteins may be due to differences in surfactant binding, as previously observed for other mutations that alter charge (Shi et al., 2008; Rath et al., 2009; De Zutter et al., 2013). Quantification of the effects of Ent5 mutations on (B) Ent5–Gga2 interaction or (C) Ent5–Chc1 interaction (n = 3). Error bars indicate SD; p values determined with Student’s t test. (D) Chc1-box mutation blocks interaction of Ent5 with clathrin. Clc1 was immunoprecipitated from lysates of cells lacking CHC1 transformed with plasmids that contain wild-type CHC1 or chc1Δbox. (E) Live-cell imaging of CHC1-deleted cells transformed with plasmids that contain wild-type CHC1 or chc1Δbox mutant. Scale bar, 5 μm.

Mentions: The extension in lifespan of Gga2 and Chc1 structures in cells lacking Ent5 suggests that Ent5 acts as more than a cargo linker. To better understand the role of Ent5 in ECT, we investigated the importance of different Ent5 domains and motifs in Ent5 function. To do this, we mutated each of the known domains and/or motifs in Ent5 (Figure 3A). ENT5 encodes an N-terminal ANTH domain. This domain is believed to bind cargo and/or lipids. To disrupt the function of the ANTH domain, we mutated several positively charged residues that are predicted to lie on the surface of the ANTH domain and are similar to residues that interact with lipids in other ANTH domains to generate a Ent5 ANTH-domain charge-reversal mutant (Ent5-CR; Ford et al., 2002; Sun et al., 2005). Ent5 also binds the γ-ear of clathrin adaptors Gga2 and AP-1. We disrupted this activity by mutating key acidic and hydrophobic residues of the highly conserved γ-ear interaction motif to generate an Ent5 adaptor-binding mutant (Ent5ΔAB; Nogi et al., 2002; Duncan et al., 2003; Mills et al., 2003). Finally, Ent5 contains a pair of clathrin box motifs that mediate interaction with clathrin in many proteins (Dell’Angelica, 2001). We disrupted clathrin binding by mutating key residues of each clathrin box to generate an Ent5 clathrin-binding mutant (Ent5ΔCB; Figure 4A). When expressed from the endogenous ENT5 locus, each of the mutant proteins was expressed at the same level as wild-type Ent5 (Supplemental Figure S1, A–C). However, each of the mutant alleles reduced the functional activity of Ent5 as assessed using the quantitative CFW assay (Figure 3B), suggesting that each activity contributes to Ent5 function in ECT.


Clathrin binding by the adaptor Ent5 promotes late stages of clathrin coat maturation.

Hung CW, Duncan MC - Mol. Biol. Cell (2016)

Clathrin is required for maximal interaction of Ent5 with Gga2 and localization of Ent5 to membranes. (A) A representative immunoprecipitation reaction. GFP-tagged Ent5 was immunoprecipitated from cell lysates, and the immunoprecipitates were probed with antibodies to Ent5, Gga2, and clathrin to monitor the effect of Ent5 mutations on the Ent5–Gga2 and Ent5–clathrin interactions. Apparent size shift in mutant proteins may be due to differences in surfactant binding, as previously observed for other mutations that alter charge (Shi et al., 2008; Rath et al., 2009; De Zutter et al., 2013). Quantification of the effects of Ent5 mutations on (B) Ent5–Gga2 interaction or (C) Ent5–Chc1 interaction (n = 3). Error bars indicate SD; p values determined with Student’s t test. (D) Chc1-box mutation blocks interaction of Ent5 with clathrin. Clc1 was immunoprecipitated from lysates of cells lacking CHC1 transformed with plasmids that contain wild-type CHC1 or chc1Δbox. (E) Live-cell imaging of CHC1-deleted cells transformed with plasmids that contain wild-type CHC1 or chc1Δbox mutant. Scale bar, 5 μm.
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Figure 4: Clathrin is required for maximal interaction of Ent5 with Gga2 and localization of Ent5 to membranes. (A) A representative immunoprecipitation reaction. GFP-tagged Ent5 was immunoprecipitated from cell lysates, and the immunoprecipitates were probed with antibodies to Ent5, Gga2, and clathrin to monitor the effect of Ent5 mutations on the Ent5–Gga2 and Ent5–clathrin interactions. Apparent size shift in mutant proteins may be due to differences in surfactant binding, as previously observed for other mutations that alter charge (Shi et al., 2008; Rath et al., 2009; De Zutter et al., 2013). Quantification of the effects of Ent5 mutations on (B) Ent5–Gga2 interaction or (C) Ent5–Chc1 interaction (n = 3). Error bars indicate SD; p values determined with Student’s t test. (D) Chc1-box mutation blocks interaction of Ent5 with clathrin. Clc1 was immunoprecipitated from lysates of cells lacking CHC1 transformed with plasmids that contain wild-type CHC1 or chc1Δbox. (E) Live-cell imaging of CHC1-deleted cells transformed with plasmids that contain wild-type CHC1 or chc1Δbox mutant. Scale bar, 5 μm.
Mentions: The extension in lifespan of Gga2 and Chc1 structures in cells lacking Ent5 suggests that Ent5 acts as more than a cargo linker. To better understand the role of Ent5 in ECT, we investigated the importance of different Ent5 domains and motifs in Ent5 function. To do this, we mutated each of the known domains and/or motifs in Ent5 (Figure 3A). ENT5 encodes an N-terminal ANTH domain. This domain is believed to bind cargo and/or lipids. To disrupt the function of the ANTH domain, we mutated several positively charged residues that are predicted to lie on the surface of the ANTH domain and are similar to residues that interact with lipids in other ANTH domains to generate a Ent5 ANTH-domain charge-reversal mutant (Ent5-CR; Ford et al., 2002; Sun et al., 2005). Ent5 also binds the γ-ear of clathrin adaptors Gga2 and AP-1. We disrupted this activity by mutating key acidic and hydrophobic residues of the highly conserved γ-ear interaction motif to generate an Ent5 adaptor-binding mutant (Ent5ΔAB; Nogi et al., 2002; Duncan et al., 2003; Mills et al., 2003). Finally, Ent5 contains a pair of clathrin box motifs that mediate interaction with clathrin in many proteins (Dell’Angelica, 2001). We disrupted clathrin binding by mutating key residues of each clathrin box to generate an Ent5 clathrin-binding mutant (Ent5ΔCB; Figure 4A). When expressed from the endogenous ENT5 locus, each of the mutant proteins was expressed at the same level as wild-type Ent5 (Supplemental Figure S1, A–C). However, each of the mutant alleles reduced the functional activity of Ent5 as assessed using the quantitative CFW assay (Figure 3B), suggesting that each activity contributes to Ent5 function in ECT.

Bottom Line: We find that the direct binding of Ent5 with clathrin is required for its role in coat behavior and cargo traffic.Surprisingly, the interaction of Ent5 with other adaptors is dispensable for coat behavior but not cargo traffic.These findings support a model in which Ent5 clathrin binding performs a mechanistic role in coat maturation, whereas Ent5 adaptor binding promotes cargo incorporation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.

No MeSH data available.


Related in: MedlinePlus