Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase.
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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Animals, Apoptosis Regulatory Proteins, Cell Cycle Proteins, Humans, Intracellular Signaling Peptides and Proteins, Nuclear Proteins, Protein Binding, Ubiquitins, Valosin Containing Protein
Adaptor Proteins, Signal Transducing, Animals, Apoptosis Regulatory Proteins, Cell Cycle Proteins, Humans, Intracellular Signaling Peptides and Proteins, Nuclear Proteins, Protein Binding, Ubiquitins, Valosin Containing Protein
Elife
Date: Dec. 03, 2021
PubMed ID: 35920641
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