Resolving the mechanism of cofactor and substrate interactions with Cdc48

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Project Summary/Abstract Determine the structural basis of cofactor and substrate interactions with Cdc48 Cdc48 (the yeast otholog of p97) is an essential AAA+ ATPase that is involved in extracting proteins throughout the cell from a vast range of locations, including protein complexes, ribosomes, chromatin, mitochondria and the ER. This study will answer how Cdc48 processes substrates and the structural changes it must undergo to do so as well as how adaptor proteins of Cdc48 modulate its activity and help to target Cdc48 to specific substrates. To investigate the mechanism of Cdc48, we will focus on a cofactor of Cdc48 named Shp1 in Saccharomyces cerevisiae. We will perform purifications of substrate bound Cdc48 using ADP⋅BeFx, an ATP analog, to trap substrates. We will use cryo-EM to visualize the binding of the substrate(s) to Cdc48 and the conformational changes that Cdc48 undergoes. This will allow us to make conclusions about how substrate binds to and is processed by Cdc48. Using the cofactor Shp1 to learn more about Cdc48 will illuminate how the Cdc48-Shp1 complex targets and processes substrates. By focusing on particles from our cryo-EM micrographs with putative Shp1 density, and using crosslinking to enrich for these particles, we aim to better visualize the interaction of Shp1 with Cdc48. This goes beyond the direct interaction of Shp1 and Cdc48 and into the mechanism of how Shp1 interacts with substrates to target them to Cdc48s central pore. However, Shp1 is not the only cofactor of Cdc48. How other cofactors work in synchrony with Cdc48 to target and process substrates remains an open area of study. From our own unpublished data, we have shown upregulation of Car2 when purifying cofactors of Cdc48. From our data, this protein does not seem to be a substrate of Cdc48 but does seem to be linked to the presence of Cdc48 in purifications. Car2 is not an established cofactor of Cdc48. However, because of the link between Cdc48 and Car2 abundance, we suspect that Car2 may interact with Cdc48 and perhaps act as a cofactor. We will use mass spectrometry and cryo-EM to investigate any interactions that these proteins may have and the function behind these potential interactions.