Abstract
The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62–ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.
Highlights
The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis
To determine the mechanisms of action of the p62, NBR1, and TAX1BP1 cargo receptors and their cooperation in the formation and degradation of ubiquitincontaining condensates, we studied their colocalization in HAP1 cells expressing endogenously tagged GFP-p62 and mScarlet-auxininducible degradation (AID)-NBR1 (Fig. 1a and Supplementary Fig. 1 b–d)
In addition to a fluorophore, NBR1 was tagged with an auxininducible degradation (AID) tag to allow its acute depletion on a protein level (Supplementary Fig. 1b)[30]
Summary
The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. P62 oligomerizes into filaments through its N-terminal PB1 domain[23,24,25] This oligomerization is required for its ability to form condensates with ubiquitinated proteins and to avidly bind the LC3/GABARAP decorated autophagosomal membrane via its LC3 interacting region (LIR) motif and the ubiquitinated cargo via its C-terminal UBA domain (Supplementary Fig. 1a)[19,20,26]. In these processes, p62 is aided by the cargo receptor NBR1. It interacts with NBR1 to recruit FIP200 and to trigger its subsequent degradation in LC3 lipidation independent autophagy[13]
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