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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.


Loss of Ent5 disrupts traffic at the TGN and/or endosomes. (A) Loss of Ent5 disrupts traffic of Chs3. Loss of Ent5 increases the sensitivity of cells lacking Chs6 to the cell wall–binding toxin CFW. The IC50 for indicated cells. Error bars indicate SD; p values were determined by Student’s t test. (B) Loss of Ent5 disrupts localization of Tlg1-mCherry. Micrographs show Z-stack projections of Gga2-GFP, expressed from its endogenous locus, and Tlg1-mCherry, expressed from its own promoter on a 2-μ plasmid in wild-type and ent5Δ cells. Arrows indicate vacuoles visible in phase-contrast image. (C) Loss of Ent5 reduces steady-state expression levels of Tlg1-mCherry. Lysates prepared from cells expressing Tlg1-mCherry from its own promoter on a 2-μ plasmid in wild-type cells and ent5Δ cells were subjected to immunoblot analysis. (D) Loss of Ent5 does not alter the fluorescence intensity of Chc1-GFP structures and increases the fluorescence intensity of Gga2-GFP. Z-stack projections of indicated cells expressing Chc1-GFP and Gga2-GFP from their endogenous loci. (E) Fluorescence intensity measurements of structures in C. Scatterplots display mean value and SEM; p values were calculated using a two-tailed Mann–Whitney U test for the  hypothesis that the medians were equal. Horizontal bars indicate median and interquartile ranges. Scale bars, 5 μm.
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Figure 1: Loss of Ent5 disrupts traffic at the TGN and/or endosomes. (A) Loss of Ent5 disrupts traffic of Chs3. Loss of Ent5 increases the sensitivity of cells lacking Chs6 to the cell wall–binding toxin CFW. The IC50 for indicated cells. Error bars indicate SD; p values were determined by Student’s t test. (B) Loss of Ent5 disrupts localization of Tlg1-mCherry. Micrographs show Z-stack projections of Gga2-GFP, expressed from its endogenous locus, and Tlg1-mCherry, expressed from its own promoter on a 2-μ plasmid in wild-type and ent5Δ cells. Arrows indicate vacuoles visible in phase-contrast image. (C) Loss of Ent5 reduces steady-state expression levels of Tlg1-mCherry. Lysates prepared from cells expressing Tlg1-mCherry from its own promoter on a 2-μ plasmid in wild-type cells and ent5Δ cells were subjected to immunoblot analysis. (D) Loss of Ent5 does not alter the fluorescence intensity of Chc1-GFP structures and increases the fluorescence intensity of Gga2-GFP. Z-stack projections of indicated cells expressing Chc1-GFP and Gga2-GFP from their endogenous loci. (E) Fluorescence intensity measurements of structures in C. Scatterplots display mean value and SEM; p values were calculated using a two-tailed Mann–Whitney U test for the hypothesis that the medians were equal. Horizontal bars indicate median and interquartile ranges. Scale bars, 5 μm.

Mentions: Previous research implicated Ent5 functions in endosomal/TGN traffic; however, it was unclear whether Ent5 was a specialized cargo-specific adaptor or played a central mechanistic role in endosomal/TGN clathrin-dependent traffic (ECT; Costaguta et al., 2006; Copic et al., 2007). To better understand its role in ECT, we tested whether the deletion of ENT5 impaired ECT, using a quantitative calcofluor white (CFW) sensitivity assay. This assay measures the fidelity of ECT by making intracellular retention of the chitin synthase Chs3 dependent on ECT. When ECT is defective, some Chs3 is found at the cell surface in cells lacking CHS6, whereas, in otherwise wild-type cells, all Chs3 is retained intracellularly when CHS6 is deleted (Valdivia et al., 2002). Cell surface Chs3 makes the cells sensitive to CFW. We found that deletion of ENT5 increased the CFW sensitivity of cells lacking CHS6 (Figure 1A). As a further test of the role of Ent5 in ECT, we examined the localization of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor Tlg1 in cells lacking Ent5. In wild-type cells, Tlg1-mCherry was found in the vacuole and in punctate structures that colocalized with Gga2-GFP, consistent with the known localization of Tlg1 at the TGN. In contrast, in cells lacking Ent5, Tlg1-mCherry puncta were rarer and dimmer than in wild-type cells (Figure 1B). Furthermore, steady-state levels of Tlg1 are lower in cells lacking Ent5, suggesting that Tlg1 is missorted to the vacuole and degraded in these cells (Figure 1C). These results are consistent with the loss of Ent5 causing a defect in ECT but do not distinguish between cargo-specific or central mechanistic roles for Ent5.


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

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

Loss of Ent5 disrupts traffic at the TGN and/or endosomes. (A) Loss of Ent5 disrupts traffic of Chs3. Loss of Ent5 increases the sensitivity of cells lacking Chs6 to the cell wall–binding toxin CFW. The IC50 for indicated cells. Error bars indicate SD; p values were determined by Student’s t test. (B) Loss of Ent5 disrupts localization of Tlg1-mCherry. Micrographs show Z-stack projections of Gga2-GFP, expressed from its endogenous locus, and Tlg1-mCherry, expressed from its own promoter on a 2-μ plasmid in wild-type and ent5Δ cells. Arrows indicate vacuoles visible in phase-contrast image. (C) Loss of Ent5 reduces steady-state expression levels of Tlg1-mCherry. Lysates prepared from cells expressing Tlg1-mCherry from its own promoter on a 2-μ plasmid in wild-type cells and ent5Δ cells were subjected to immunoblot analysis. (D) Loss of Ent5 does not alter the fluorescence intensity of Chc1-GFP structures and increases the fluorescence intensity of Gga2-GFP. Z-stack projections of indicated cells expressing Chc1-GFP and Gga2-GFP from their endogenous loci. (E) Fluorescence intensity measurements of structures in C. Scatterplots display mean value and SEM; p values were calculated using a two-tailed Mann–Whitney U test for the  hypothesis that the medians were equal. Horizontal bars indicate median and interquartile ranges. Scale bars, 5 μm.
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Related In: Results  -  Collection

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Figure 1: Loss of Ent5 disrupts traffic at the TGN and/or endosomes. (A) Loss of Ent5 disrupts traffic of Chs3. Loss of Ent5 increases the sensitivity of cells lacking Chs6 to the cell wall–binding toxin CFW. The IC50 for indicated cells. Error bars indicate SD; p values were determined by Student’s t test. (B) Loss of Ent5 disrupts localization of Tlg1-mCherry. Micrographs show Z-stack projections of Gga2-GFP, expressed from its endogenous locus, and Tlg1-mCherry, expressed from its own promoter on a 2-μ plasmid in wild-type and ent5Δ cells. Arrows indicate vacuoles visible in phase-contrast image. (C) Loss of Ent5 reduces steady-state expression levels of Tlg1-mCherry. Lysates prepared from cells expressing Tlg1-mCherry from its own promoter on a 2-μ plasmid in wild-type cells and ent5Δ cells were subjected to immunoblot analysis. (D) Loss of Ent5 does not alter the fluorescence intensity of Chc1-GFP structures and increases the fluorescence intensity of Gga2-GFP. Z-stack projections of indicated cells expressing Chc1-GFP and Gga2-GFP from their endogenous loci. (E) Fluorescence intensity measurements of structures in C. Scatterplots display mean value and SEM; p values were calculated using a two-tailed Mann–Whitney U test for the hypothesis that the medians were equal. Horizontal bars indicate median and interquartile ranges. Scale bars, 5 μm.
Mentions: Previous research implicated Ent5 functions in endosomal/TGN traffic; however, it was unclear whether Ent5 was a specialized cargo-specific adaptor or played a central mechanistic role in endosomal/TGN clathrin-dependent traffic (ECT; Costaguta et al., 2006; Copic et al., 2007). To better understand its role in ECT, we tested whether the deletion of ENT5 impaired ECT, using a quantitative calcofluor white (CFW) sensitivity assay. This assay measures the fidelity of ECT by making intracellular retention of the chitin synthase Chs3 dependent on ECT. When ECT is defective, some Chs3 is found at the cell surface in cells lacking CHS6, whereas, in otherwise wild-type cells, all Chs3 is retained intracellularly when CHS6 is deleted (Valdivia et al., 2002). Cell surface Chs3 makes the cells sensitive to CFW. We found that deletion of ENT5 increased the CFW sensitivity of cells lacking CHS6 (Figure 1A). As a further test of the role of Ent5 in ECT, we examined the localization of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor Tlg1 in cells lacking Ent5. In wild-type cells, Tlg1-mCherry was found in the vacuole and in punctate structures that colocalized with Gga2-GFP, consistent with the known localization of Tlg1 at the TGN. In contrast, in cells lacking Ent5, Tlg1-mCherry puncta were rarer and dimmer than in wild-type cells (Figure 1B). Furthermore, steady-state levels of Tlg1 are lower in cells lacking Ent5, suggesting that Tlg1 is missorted to the vacuole and degraded in these cells (Figure 1C). These results are consistent with the loss of Ent5 causing a defect in ECT but do not distinguish between cargo-specific or central mechanistic roles for Ent5.

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.