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Title: FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates

Abstract

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326–380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the “Claw” for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH); European Research Council (ERC); Austrian Science Fund (FWF)
OSTI Identifier:
1547657
Alternate Identifier(s):
OSTI ID: 1623701; OSTI ID: 1650057
Grant/Contract Number:  
AC02-05CH11231; 646653; 616024; P30401-B21; W1261; R01 GM111730; R01 GM124149; P30 GM124169
Resource Type:
Published Article
Journal Name:
Molecular Cell
Additional Journal Information:
Journal Name: Molecular Cell Journal Volume: 74 Journal Issue: 2; Journal ID: ISSN 1097-2765
Publisher:
Cell Press - Elsevier
Country of Publication:
United States
Language:
English
Subject:
Biochemistry & Molecular Biology; Cell Biology; selective autophagy; phase separation; ubiquitin; X-ray crystallography; biochemistry; cell biology; ATG8; quality control; 59 BASIC BIOLOGICAL SCIENCES; x-ray crystallography

Citation Formats

Turco, Eleonora, Witt, Marie, Abert, Christine, Bock-Bierbaum, Tobias, Su, Ming-Yuan, Trapannone, Riccardo, Sztacho, Martin, Danieli, Alberto, Shi, Xiaoshan, Zaffagnini, Gabriele, Gamper, Annamaria, Schuschnig, Martina, Fracchiolla, Dorotea, Bernklau, Daniel, Romanov, Julia, Hartl, Markus, Hurley, James H., Daumke, Oliver, and Martens, Sascha. FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates. United States: N. p., 2019. Web. doi:10.1016/j.molcel.2019.01.035.
Turco, Eleonora, Witt, Marie, Abert, Christine, Bock-Bierbaum, Tobias, Su, Ming-Yuan, Trapannone, Riccardo, Sztacho, Martin, Danieli, Alberto, Shi, Xiaoshan, Zaffagnini, Gabriele, Gamper, Annamaria, Schuschnig, Martina, Fracchiolla, Dorotea, Bernklau, Daniel, Romanov, Julia, Hartl, Markus, Hurley, James H., Daumke, Oliver, & Martens, Sascha. FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates. United States. https://doi.org/10.1016/j.molcel.2019.01.035
Turco, Eleonora, Witt, Marie, Abert, Christine, Bock-Bierbaum, Tobias, Su, Ming-Yuan, Trapannone, Riccardo, Sztacho, Martin, Danieli, Alberto, Shi, Xiaoshan, Zaffagnini, Gabriele, Gamper, Annamaria, Schuschnig, Martina, Fracchiolla, Dorotea, Bernklau, Daniel, Romanov, Julia, Hartl, Markus, Hurley, James H., Daumke, Oliver, and Martens, Sascha. Mon . "FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates". United States. https://doi.org/10.1016/j.molcel.2019.01.035.
@article{osti_1547657,
title = {FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates},
author = {Turco, Eleonora and Witt, Marie and Abert, Christine and Bock-Bierbaum, Tobias and Su, Ming-Yuan and Trapannone, Riccardo and Sztacho, Martin and Danieli, Alberto and Shi, Xiaoshan and Zaffagnini, Gabriele and Gamper, Annamaria and Schuschnig, Martina and Fracchiolla, Dorotea and Bernklau, Daniel and Romanov, Julia and Hartl, Markus and Hurley, James H. and Daumke, Oliver and Martens, Sascha},
abstractNote = {The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326–380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the “Claw” for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation.},
doi = {10.1016/j.molcel.2019.01.035},
journal = {Molecular Cell},
number = 2,
volume = 74,
place = {United States},
year = {2019},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1016/j.molcel.2019.01.035

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Cited by: 37 works
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Figures / Tables:

Figure-1 Figure-1: The FIP200 C-Terminal Region Directly Interacts with p62 in a LIR-Dependent Manner (A) Schematic representation of FIP200 and p62. The Atg11 homology domain of FIP200 is depicted in gray. The FIP200-interacting region (FIR) of p62 is depicted in pink and the LIR motif in dark gray. p62 constructs coveringmore » parts of the FIR are shown on the right. (B) GST or GST-FIP200 CTR were coupled to glutathione (GSH) beads and incubated with HeLa cells lysates (200 μg). Beads were washed, and the beads/flow-through fractions were analyzed by western blot using anti-p62 antibody. The bound sample was probed with anti-GST to visualize the amount of bait protein on the beads. (C) GSH beads were coated with GST or GST-FIP200 CTR and incubated with recombinant mCherry-p62 (2 μM). Beads were imaged by microscopy. (D) The experimental setup is shown on the left. GSH beads were coated with GST-2x ubiquitin. Excess GST-2x ubiquitin was washed off, and beads were incubated with mCherry-p62 (2 μM) and GFP-FIP200 CTR aa 1429–2594 (5 μM). After 1 h incubation, beads were imaged by microscopy. (E) GSH beads were coated with the indicated GST-p62 constructs and incubated with GFP-FIP200 CTR aa 1429–1594 (5 μM). Beads at equilibrium were imaged by microscopy. Protein inputs are shown in Figure S1B. The GFP signal on the beads was normalized to the signal of GFP-FIP200 CTR bound to GST-p62 FIR-coated beads. Average intensity and SEM for n = 3 are plotted. Significant differences are indicated with when p value ≤ 0.05, ∗∗ when p value ≤ 0.01, and ∗∗∗ when p value ≤ 0.001. (F) Same experimental setup and quantification used in (E). Beads were imaged by microscopy. Protein inputs are shown in Figure S1C. The GST-p62 FIR-LIRmut construct carries a mutation in the LIR motif (335DDDW338 > AAAA). (G) Same experimental setup used in (C). In the mCherry p62 LIRmut residues 335-338 are mutated to Ala. Protein inputs are shown in Figure S1D. The mCherry signal on the beads was normalized to the signal of mCherry-p62 WT on GST-FIP200 CTR-coated beads. The average intensity and SEM for n = 3 are shown. Significant differences are indicated with when p value ≤ 0.05, ∗∗ when p value ≤ 0.01, and ∗∗∗ when p value ≤ 0.001. (H) The same experimental setup used in (E) and (F) was used to test the interaction of the Atg19 C terminus (Atg19 C-ter) with the FIP200 CTR. In the Atg19 C-ter-mut, the three LIR motifs (Figure S1F) are mutated. The graph on the bottom shows the average GFP intensity, normalized to the signal of GFP-FIP200 CTR binding to GST-Atg19 C-ter, and SEM for n = 3. Significant differences are indicated with when p value ≤ 0.05, ∗∗ when p value ≤ 0.01, and ∗∗∗ when p value ≤ 0.001. Protein inputs are shown in Figure S1E.« less

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