Article

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating

Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2006; 103(27):10265-70. DOI: 10.1073/pnas.0603369103
Source: PubMed

ABSTRACT

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

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Available from: Ramiro Horacio Massol
    • "In turn, the newly generated PtdIns(3,4)P2 recruits the PX-BAR domain protein Sorting nexin-9 and possibly other effectors to mature clathrincoated pits just prior to dynamin-mediated fission (Posor et al., 2013 ). Conversion of PtdIns(4,5)P2 into PtdIns(3,4)P2 by the successive action of Synaptojanin-1 (which forms PtdIns(4)P) and PI3K-C2-alpha (which uses PtdIns(4)P) might also contribute to clathrin coat removal given that the uncoating factor auxilin is recruited to membranes by PtdIns3P, PtdIns4P, or PtdIns(3,4)P2 (Massol et al., 2006 ). Moreover, these PtdInsP metabolizing enzymes modify the lipid composition of the vesicle membrane in a way that confers an ''endosome-like identity,'' which might allow fusion with an endosomal intermediate compartment (Jä hne et al., 2015). "
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    • "Reagents, cell culture, and transfections U373 human glioblastoma cells stably expressing σ2-EGFP were grown in DMEM containing 10% fetal calf serum (FCS), penicillin, and streptomycin. Transient expression of rat Tomato-LCa (Massol et al., 2006; Saffarian et al., 2009) and LifeAct-mCherry was carried out by transfection with Lipofectamine 2000 (Invitrogen) in Optimem (Life Technologies, Grand Island, NY) according to the manufacturers' instructions. SUM159 human breast carcinoma cells were geneedited to replace both alleles of the σ2 subunit of AP2 with σ2 fused at its C-terminus to EGFP using a transcription activator-like effector nuclease (TALEN)-based protocol (Sanjana et al., 2012; Cocucci et al., 2014). "
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    • "Thus, auxilin functions as an adaptor that attracts Hsc70 to the clathrin coat. The precise regulation of irreversible uncoating by Hsc70 is unclear, but from life-cell imaging it has been inferred that coat disassembly is preceded in the cell by the budding of coated membranes [36,37]. The methods described above have now allowed us to test whether a spherical lattice is required for irreversible uncoating or if in principle clathrin-clathrin interactions could also be disrupted within planar arrangements by Hsc70. "
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