Journal: Nature structural & molecular biology
Article Title: Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4
Figure Lengend Snippet: Functional analysis of Zn 2+ -finger and MPN domain mutations a , Unfolding of poly-ubiquitinated Eos by wild-type S. cerevisiae . b, A npl4-1 temperature-sensitive S. cerevisiae strain was transformed with a plasmid encoding wild-type Npl4 or the indicated Zn 2+ -finger mutants, spotted in serial dilution, and incubated at the indicated temperatures for two days (30 and 37°C) or three days (25°C). c , As in a . d , Binding of poly-ubiquitinated substrate to SBP-tagged C. thermophilum Npl4 (Zn 2+ -finger/MPN/CTD domains, residues 129-602) or the indicated variants (MPN only: residues 129-519, CTD only: residues 519-602). The bait proteins were bound to streptavidin beads and incubated with dye-labeled, poly-ubiquitinated superfolder GFP. Bound material was analyzed by SDS-PAGE followed by fluorescence scanning (top) and Coomassie blue staining (bottom). M, molecular weight markers. e , The locations of MPN cleft mutants tested in d are shown in stick representation. The MPN, insert-1, insert-2, and CTD are shown in tan, magenta, purple, and orange, respectively.
Article Snippet: The C. thermophilum Npl4 Zn2+ -finger/MPN/CTD fragments (residues 129-602, 129-519, or 519-602) with an N-terminal SBP tag were incubated with streptavidin agarose beads (Pierce) in binding buffer (50 mM Tris pH 8.0, 150 mM NaCl) for 30 min at room temperature.
Techniques: Functional Assay, Transformation Assay, Plasmid Preparation, Serial Dilution, Incubation, Binding Assay, Labeling, SDS Page, Fluorescence, Staining, Molecular Weight