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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.


Model of coat assembly. 1) Gga2 accumulates on membranes and recruits clathrin. 2) Ent5, initially recruited via its ANTH domain, is stabilized by interaction with clathrin and Gga2. This allows the recruitment of Ent5-specific cargo. 3) Ent5 stimulates the formation of the transport carrier, possibly by inducing clathrin polymerization or forming a subclathrin coat. Gga2 helps to increase the amount of Ent5 in the carrier either through initial recruitment of more Ent5 or, as illustrated, by Gga2 indirectly linking Ent5 to the clathrin coat.
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Figure 6: Model of coat assembly. 1) Gga2 accumulates on membranes and recruits clathrin. 2) Ent5, initially recruited via its ANTH domain, is stabilized by interaction with clathrin and Gga2. This allows the recruitment of Ent5-specific cargo. 3) Ent5 stimulates the formation of the transport carrier, possibly by inducing clathrin polymerization or forming a subclathrin coat. Gga2 helps to increase the amount of Ent5 in the carrier either through initial recruitment of more Ent5 or, as illustrated, by Gga2 indirectly linking Ent5 to the clathrin coat.

Mentions: We propose that the key function performed by Ent5 is the stimulation of clathrin assembly (Figure 6). This is consistent with the extended lifespan of coats containing Ent5, Gga2, and clathrin in cells expressing Ent5-CB. In contrast, despite reduced Ent5 recruitment, Ent5-AB–containing coats do not stall. We speculate that in wild-type and in Ent5-AB–expressing cells, Ent5 binding to clathrin promotes a conformational organization of clathrin that enables clathrin polymerization. This suggests that Gga2 on its own is not capable of promoting this conformational organization. This may be because Gga2 contains only one copy of the clathrin- binding motif known as a clathrin box (Drake and Traub, 2001). A single clathrin box can bind clathrin; however, two clathrin boxes appear to be required for clathrin assembly in vitro (Holkar et al., 2015). Although Gga2 contains many clathrin-binding sites in addition to the clathrin box, these additional sites are not clathrin boxes. They are low-affinity DLL-type binding sites, which may not promote rapid clathrin polymerization (Dannhauser et al., 2015; Zhuo et al., 2015). Thus assembly may rely on Ent5 alone, which contains two clathrin boxes, or on the cooperation of the three clathrin boxes available when both Ent5 and Gga2 are present. Our findings are similar to a recent study showing that acute inhibition of EpsinR, the closest orthologue of Ent5 in mammalian cells, inhibits clathrin coat assembly at a stage after clathrin recruitment but before disassembly (Hirst et al., 2015). However, these results and ours are also consistent with other models, such as a role for Ent5 and EpsinR in disassembling clathrin coats. Therefore resolving the exact molecular mechanism by which Ent5 and EpsinR interfere with traffic will require an in vitro system that monitors assembly and organization directly using these proteins.


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

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

Model of coat assembly. 1) Gga2 accumulates on membranes and recruits clathrin. 2) Ent5, initially recruited via its ANTH domain, is stabilized by interaction with clathrin and Gga2. This allows the recruitment of Ent5-specific cargo. 3) Ent5 stimulates the formation of the transport carrier, possibly by inducing clathrin polymerization or forming a subclathrin coat. Gga2 helps to increase the amount of Ent5 in the carrier either through initial recruitment of more Ent5 or, as illustrated, by Gga2 indirectly linking Ent5 to the clathrin coat.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: Model of coat assembly. 1) Gga2 accumulates on membranes and recruits clathrin. 2) Ent5, initially recruited via its ANTH domain, is stabilized by interaction with clathrin and Gga2. This allows the recruitment of Ent5-specific cargo. 3) Ent5 stimulates the formation of the transport carrier, possibly by inducing clathrin polymerization or forming a subclathrin coat. Gga2 helps to increase the amount of Ent5 in the carrier either through initial recruitment of more Ent5 or, as illustrated, by Gga2 indirectly linking Ent5 to the clathrin coat.
Mentions: We propose that the key function performed by Ent5 is the stimulation of clathrin assembly (Figure 6). This is consistent with the extended lifespan of coats containing Ent5, Gga2, and clathrin in cells expressing Ent5-CB. In contrast, despite reduced Ent5 recruitment, Ent5-AB–containing coats do not stall. We speculate that in wild-type and in Ent5-AB–expressing cells, Ent5 binding to clathrin promotes a conformational organization of clathrin that enables clathrin polymerization. This suggests that Gga2 on its own is not capable of promoting this conformational organization. This may be because Gga2 contains only one copy of the clathrin- binding motif known as a clathrin box (Drake and Traub, 2001). A single clathrin box can bind clathrin; however, two clathrin boxes appear to be required for clathrin assembly in vitro (Holkar et al., 2015). Although Gga2 contains many clathrin-binding sites in addition to the clathrin box, these additional sites are not clathrin boxes. They are low-affinity DLL-type binding sites, which may not promote rapid clathrin polymerization (Dannhauser et al., 2015; Zhuo et al., 2015). Thus assembly may rely on Ent5 alone, which contains two clathrin boxes, or on the cooperation of the three clathrin boxes available when both Ent5 and Gga2 are present. Our findings are similar to a recent study showing that acute inhibition of EpsinR, the closest orthologue of Ent5 in mammalian cells, inhibits clathrin coat assembly at a stage after clathrin recruitment but before disassembly (Hirst et al., 2015). However, these results and ours are also consistent with other models, such as a role for Ent5 and EpsinR in disassembling clathrin coats. Therefore resolving the exact molecular mechanism by which Ent5 and EpsinR interfere with traffic will require an in vitro system that monitors assembly and organization directly using these proteins.

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.