Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

Labib, K.; Fujisawa, R.

doi:10.1101/2022.01.13.476277

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bioRxiv · 10.1101/2022.01.13.476277

Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

Labib, K.·Fujisawa, R.

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Abstract

The unfolding of ubiquitylated proteins by the p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 is essential in many areas of eukaryotic cell biology. Previous studies showed that yeast Cdc48-Ufd1-Npl4 is governed by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that substrate processing by mammalian p97-UFD1-NPL4 involves a complex interplay between ubiquitin chain length and additional p97 cofactors. Using disassembly of the ubiquitylated CMG helicase as a model in vitro system, we find that reconstituted p97-UFD1-NPL4 only unfolds substrates with very long ubiquitin chains. However, this high ubiquitin threshold is greatly reduced, to a level resembling yeast Cdc48-Ufd1-Npl4, by the UBXN7, FAF1 or FAF2 partners of mammalian p97-UFD1-NPL4. Stimulation by UBXN7/FAF1/FAF2 requires the UBX domain that connects each factor to p97, together with the ubiquitin-binding UBA domain of UBXN7 and a previously uncharacterised coiled-coil domain in FAF1/FAF2. Furthermore, we show that deletion of the UBXN7 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.

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Labib, K., Fujisawa, R.. 2022-01-14. Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase. https://doi.org/10.1101/2022.01.13.476277

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