Limits...
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.


Schematic of Ent5 and mutations generated. (A) Top, ANTH domain is indicated in gray, charged patches predicted to be important for ANTH function are indicated as CP1 and CP2, adaptor-binding sites are indicated as AB1 and AB2, and clathrin boxes are indicated as CB1 and CB2. Bottom, residues mutated are underlined. For ent5-CR, alignment with rat AP180 and CALM is shown. Residues important for phosphoinositide-binding residues in AP180 and CALM are red; underlined residues were mutated to glutamic acid. For ent5ΔAB and ent5ΔCB alignment, the consensus sequence is shown (cons); underlined residues were mutated to alanine. (B) CFW sensitivity of indicated mutants. p = 0.0034 for chs6Δ vs. ent5Δ, p = 0.0004 for chs6Δ vs. LB, p = 0.0374 for chs6Δ vs. AB, p = 0.0174 for chs6Δ vs. CB, p = 0.0005 for chs6Δ vs. ABCB, and p < 0.0001 for chs6Δ vs. wild type. Error bars indicate SD; p values were determined using Student’s t test. (C) Z-stack projection of Ent5-GFP and Chc1-RFP in indicated mutants. (D) Quantification of the number of Ent5-GFP puncta per cell in a central plane. (E) Fluorescence intensity measurements of individual puncta in indicated cells. Scatterplots display mean value and SEM; horizontal bars indicate median and interquartile ranges. Scale bar, 5 μm. p values reflect a two-tailed Mann–Whitney test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4814221&req=5

Figure 3: Schematic of Ent5 and mutations generated. (A) Top, ANTH domain is indicated in gray, charged patches predicted to be important for ANTH function are indicated as CP1 and CP2, adaptor-binding sites are indicated as AB1 and AB2, and clathrin boxes are indicated as CB1 and CB2. Bottom, residues mutated are underlined. For ent5-CR, alignment with rat AP180 and CALM is shown. Residues important for phosphoinositide-binding residues in AP180 and CALM are red; underlined residues were mutated to glutamic acid. For ent5ΔAB and ent5ΔCB alignment, the consensus sequence is shown (cons); underlined residues were mutated to alanine. (B) CFW sensitivity of indicated mutants. p = 0.0034 for chs6Δ vs. ent5Δ, p = 0.0004 for chs6Δ vs. LB, p = 0.0374 for chs6Δ vs. AB, p = 0.0174 for chs6Δ vs. CB, p = 0.0005 for chs6Δ vs. ABCB, and p < 0.0001 for chs6Δ vs. wild type. Error bars indicate SD; p values were determined using Student’s t test. (C) Z-stack projection of Ent5-GFP and Chc1-RFP in indicated mutants. (D) Quantification of the number of Ent5-GFP puncta per cell in a central plane. (E) Fluorescence intensity measurements of individual puncta in indicated cells. Scatterplots display mean value and SEM; horizontal bars indicate median and interquartile ranges. Scale bar, 5 μm. p values reflect a two-tailed Mann–Whitney test.

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)

Schematic of Ent5 and mutations generated. (A) Top, ANTH domain is indicated in gray, charged patches predicted to be important for ANTH function are indicated as CP1 and CP2, adaptor-binding sites are indicated as AB1 and AB2, and clathrin boxes are indicated as CB1 and CB2. Bottom, residues mutated are underlined. For ent5-CR, alignment with rat AP180 and CALM is shown. Residues important for phosphoinositide-binding residues in AP180 and CALM are red; underlined residues were mutated to glutamic acid. For ent5ΔAB and ent5ΔCB alignment, the consensus sequence is shown (cons); underlined residues were mutated to alanine. (B) CFW sensitivity of indicated mutants. p = 0.0034 for chs6Δ vs. ent5Δ, p = 0.0004 for chs6Δ vs. LB, p = 0.0374 for chs6Δ vs. AB, p = 0.0174 for chs6Δ vs. CB, p = 0.0005 for chs6Δ vs. ABCB, and p < 0.0001 for chs6Δ vs. wild type. Error bars indicate SD; p values were determined using Student’s t test. (C) Z-stack projection of Ent5-GFP and Chc1-RFP in indicated mutants. (D) Quantification of the number of Ent5-GFP puncta per cell in a central plane. (E) Fluorescence intensity measurements of individual puncta in indicated cells. Scatterplots display mean value and SEM; horizontal bars indicate median and interquartile ranges. Scale bar, 5 μm. p values reflect a two-tailed Mann–Whitney test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814221&req=5

Figure 3: Schematic of Ent5 and mutations generated. (A) Top, ANTH domain is indicated in gray, charged patches predicted to be important for ANTH function are indicated as CP1 and CP2, adaptor-binding sites are indicated as AB1 and AB2, and clathrin boxes are indicated as CB1 and CB2. Bottom, residues mutated are underlined. For ent5-CR, alignment with rat AP180 and CALM is shown. Residues important for phosphoinositide-binding residues in AP180 and CALM are red; underlined residues were mutated to glutamic acid. For ent5ΔAB and ent5ΔCB alignment, the consensus sequence is shown (cons); underlined residues were mutated to alanine. (B) CFW sensitivity of indicated mutants. p = 0.0034 for chs6Δ vs. ent5Δ, p = 0.0004 for chs6Δ vs. LB, p = 0.0374 for chs6Δ vs. AB, p = 0.0174 for chs6Δ vs. CB, p = 0.0005 for chs6Δ vs. ABCB, and p < 0.0001 for chs6Δ vs. wild type. Error bars indicate SD; p values were determined using Student’s t test. (C) Z-stack projection of Ent5-GFP and Chc1-RFP in indicated mutants. (D) Quantification of the number of Ent5-GFP puncta per cell in a central plane. (E) Fluorescence intensity measurements of individual puncta in indicated cells. Scatterplots display mean value and SEM; horizontal bars indicate median and interquartile ranges. Scale bar, 5 μm. p values reflect a two-tailed Mann–Whitney test.
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.