FAF1 cofactor enhances UFD1/NPL4-p97 unfolding efficiency across ubiquitin chain lengths independent of SUMO2

The AAA+ protein p97/VCP and its cofactor UFD1/NPL4 interact with and unfold ubiquitinated proteins to promote disaggregation and unfolding for recycling or to prepare substrates for proteasomal degradation. The cofactor Fas-associated factor 1 (FAF1) is suggested to reduce the length of ubiquitin chain required for substrate unfolding by UFD1/NPL4-p97 and to interact with SUMO. Here, we employ in vitro reconstitution of UFD1/NPL4-p97 and FAF1/UFD1/NPL4-p97 complexes and fluorescent substrates modified with SUMO2-polyubiquitin hybrid or polyubiquitin-only chains of varying lengths to assess initial rates of unfolding. These assays reveal that FAF1 enhances initial rates of unfolding relative to UFD1/NPL4-p97 in a manner that is independent of SUMO2 and semi-dependent on ubiquitin chain length. Unlike preferences observed for yeast Ufd1/Npl4-Cdc48, these data suggest that the FAF1 cofactor does not contribute to preferential unfolding of the SUMO2-polyubiquitin substrates tested. Further dissection of FAF1 reveals that it significantly increases the rate of unfolding for all ubiquitin chain lengths tested with its greatest differential impact observed when unfolding chains with four to ten ubiquitin molecules that are considered physiologically relevant. Using cryoEM we resolve a series of reconstructions that reveal FAF1/UFD1/NPL4-p97 bound to substrate in non-translocating and translocating states. Observed interactions between a helix of FAF1 and UFD1 throughout the unfolding process are consistent with AlphaFold models and recent reports suggesting that FAF1 may stabilize interactions between UFD1, NPL4, and p97 to promote substrate engagement and unfolding.