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Molecular function of SEP domain adapters in VCP/p97 mediated protein unfolding

The ATPase p97 (also known as VCP) is a member of family of AAA+ ATPases, a group of enzymes that form hexameric rings and unfold substrate proteins by ATP powered translocation through the central channel. The protein is highly conserved in metazoans, with homologues found in yeast (Cdc48) and flies (Ter94) and represents up to 1% of the total protein mass in the cell. Its main function is the extraction and subsequent unfolding of mostly ubiquitinated substrate proteins from membranes and protein complexes. In this capacity, p97 is an important factor in many cellular processes, including proteasomal degradation of misfolded proteins, clearance of cellular organelles via autophagy, cell cycle regulation and DNA repair. Substrate specificity is controlled by adapter proteins and cofactors that control substrate recruitment and p97 activity. Although up to 30 different proteins that interact with p97 in this manner have been identified, the exact mechanism by which these adapters facilitate substrate processing by p97 is only sufficiently understood for the Ufd1-Npl4 pair, which is the major adapter for polyubiquitinated substrates. Recent discoveries had identified the SDS22-PP1-I3 (SPI) complex, which forms during biogenesis of the phosphatase PP1, as a new substrate, whose disassembly by p97 together with the adapter p37 was not dependent on ubiquitination. This thesis focused on the elucidation of the mechanism by which adapter proteins from the family of SEP domain containing proteins (p37, p47, UBXN2A, UBXN11) facilitate this process. We established a new in vitro fluorescence-unfolding assay that allowed us to determine that I3 is being unfolded by p97 during SPI complex disassembly. Further investigation on the function of p37 and the differences with the other three SEP domain proteins revealed that only UBXN2A was equally capable to support the unfolding reaction, while p97 remained inactive in the presence of p47 or UBXN11. Systematic exchange of protein domains between p37 and p47 showed that the inability of p47 to support unfolding was caused by a divergence in the sequence of a C-terminal linker region, which was found to be critical for binding of the SPI complex by p37. Furthermore, p47 impeded unfolding through inhibition of the ATPase activity of p97 by a “brake” motif in its N-terminal region. Additional investigations into the N-terminal structure of p37 led to the identification of an amphipathic helix that was important for efficient unfolding. Using genetically encoded crosslinkers and mass spectrometry we could show that p37 recruits the SPI complex through a multivalent interface, which includes the N-terminal helix, the SEP domain and the C-terminal linker. This interaction involves binding of I3 by the N-terminus and the SEP domain of p37 close to the pore of p97, while the C-terminal linker of p37 binds to PP1 and positions at a distance from the pore. This primes I3 as the unfolding substrate of p97 and prevents unfolding of PP1. These results demonstrate the mechanism by which the SEP domain adapters direct unfolding of a non-ubiquitinated substrate of p97.