Skip to main content
. 2006 Mar;17(3):1075–1084. doi: 10.1091/mbc.E05-04-0375

Figure 1.

Figure 1.

Direct binding of WldS/N70 sequence to VCP. (A) The N-terminal 70 amino acids of murine Ube4b (N70) are incorporated into the neuroprotective, chimeric WldS protein, fused in-frame to the entire coding sequence (285 amino acids) of Nmnat1 and an 18-amino acid linking region. In wild-type Ube4b, N70 forms part of a 123-amino acid sequence that is essential for ubiquitination activity. (B) A protein of ca. 97 kDa was pulled down from wild-type mouse brain by GST-WldS and by GST-N70. Bottom panel, a Coomassie-stained gel of the amounts of each protein used for pulldown, except that the GST sample has been diluted 1:10. The larger N70 and WldS proteins were obtained in lower quantities but were still able to precipitate significant amounts of the 97-kDa protein. (C) Tryptic peptides of VCP were identified by MALDI-TOF. Peptides marked with an asterisk (*), plus three additional VCP peptides (unpublished results), were present both in the protein pulled down by N70 and in the one pulled down by WldS. (D) Coimmunoprecipitation of VCP with WldS protein. Antibody Wld-18 was able to immunoprecipitate WldS protein (top) from nuclear extracts of mutant mouse brain (right) but not from wild-type mouse brain (left). Coimmunoprecipitation of VCP showed the same pattern (middle), whereas control protein NeuN is not coimmunoprecipitated (bottom) showing that the VCP result is specific. (E) Evidence that VCP binds directly to the N-terminus of WldS and Ube4b. Left, Coomassie-stained gel showing purified VCP and its precipitation by WldS peptides; right, Western blot confirming identity of the precipitated VCP and its complete absence in control lanes. The N-terminal 23 amino acids (N23) fused to GST (see also Figure 3) are able to precipitate purified His-tagged VCP. Neither GST alone, nor amino acids 47–70 possess this specific binding activity. (F) VCP Western blot of wild-type and WldS mouse brain homogenates showing that steady state levels of VCP are not altered by the presence of WldS. Below: Coomassie stain showing equal loading. D–F are representative of at least two experiments.