Mechanism of cargo-directed Atg8 conjugation during selective autophagy
- University of Vienna, Austria
Abstract
Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12∼Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12∼Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12∼Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.
Article and author information
Author details
Funding
European Research Council (260304)
- Sascha Martens
European Research Council (646653)
- Sascha Martens
European Research Council (279408)
- Bojan Zagrovic
Austrian Science Fund (P25546-B20)
- Sascha Martens
Austrian Science Fund (P25522-B20)
- Claudine Kraft
Austrian Science Fund (P28113-B28)
- Claudine Kraft
Austrian Science Fund (T724-B20)
- Justyna Sawa-Makarska
Vienna Science and Technology Fund (VRG10-791 001)
- Claudine Kraft
European Molecular Biology Organization (YIP)
- Claudine Kraft
- Sascha Martens
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Hong Zhang, Institute of Biophysics, Chinese Academy of Sciences, China
Publication history
- Received: June 6, 2016
- Accepted: November 21, 2016
- Accepted Manuscript published: November 23, 2016 (version 1)
- Version of Record published: December 8, 2016 (version 2)
Copyright
© 2016, Fracchiolla et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,013
- Page views
-
- 1,010
- Downloads
-
- 44
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Mechanism of cargo-directed Atg8 conjugation during selective autophagy
eLife 5:e18544.
https://doi.org/10.7554/eLife.18544
Further reading
-
- Biochemistry and Chemical Biology
- Chromosomes and Gene Expression
The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt's activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu, Viennet, et al, 2020). Here we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver.
