FIGURE 4.

Effects of mutating the Cdc48-binding site in the PUL domain of Doa1. A, expanded view of the p97 binding pocket in the PUL domain of PLAA. Conserved residues are colored in purple (top panel) with the residues targeted for mutagenesis to make the doa1^ΔCdc48 mutant in red (bottom panel). B, recombinant epitope-tagged PUL domains of Doa1 and Doa1^ΔCdc48 were subjected to binding studies with GST alone (ø) or GST-Cdc48 C terminus. Bound fractions were immunoblotted with a 50% equivalent of input lysate. C, immunoblots from doa1Δ cells transformed with vector, wild type (WT) DOA1, or plasmid expressing Doa1^ΔCdc48. Cell lysates were blotted for Doa1-V5 (anti-V5 antibody) or 3-phosphoglycerate. Stained membrane is shown as loading control. D, immunoblots from doa1Δ cells transformed with the indicated plasmids using anti-Ub antibodies or anti-3-phosphoglycerate antibodies.