Figure 3.

Biochemical characterization of Npl4p localization and interactions. (A) Npl4p is found in both soluble and membrane-bound fractions. Equal protein from crude whole cell extract (lane 1), purified microsomes (lane 2), and ER/nuclear membrane-cleared extract (lane 3) derived from a wild-type yeast strain was separated by SDS-PAGE and Western blotted with anti-Npl4 antibodies. The band corresponding to Npl4p is indicated, and asterisks denote unidentified cross-reacting proteins. (B) The microsomal fraction of Npl4p is tightly membrane associated. Purified microsomes from a wild-type yeast strain were washed with either buffer alone (lanes 1 and 2), 1 M KOAc (lanes 3 and 4), 2 M KOAc (lanes 5 and 6), 3 M urea (lanes 7 and 8), 0.1 M Na₂CO₃, pH 11 (lanes 9 and 10), or 1% Triton X-100 (lanes 11 and 12). The samples were then separated into pellet (P; membrane associated; odd lanes) or soluble (S; even lanes) fractions and subjected to SDS-PAGE and Western blotting with anti-Npl4 and anti-Cdc48 antibodies. The extraction profiles of the peripheral ER membrane protein Sec17p and the ER integral membrane protein Sec62p were determined by Western blotting with anti-Sec17 and anti-Sec62 antibodies as controls. (C) Npl4p-protein A (pA) copurifies with Cdc48p and Ufd1p. Yeast whole cell extracts derived from either a strain expressing untagged Npl4p (lane 1) or Npl4p-pA (lane 2) were incubated with IgG-Sepharose beads. After extensive washing, proteins bound to the beads were eluted with acid and visualized by SDS-PAGE and Coomassie blue staining. Mass spectral analysis of excised protein bands revealed the identity of the Npl4p-pA copurifying p100 and p43 proteins as Cdc48p and Ufd1p, respectively.