Figure 4.
Mzt1 Stabilizes Alp6 in an “Interaction-Competent” State within Higher-Order Complexes
(A) SDS-PAGE of amylose (MBP) purification of coexpressed γ-TuSCAlp6-MBP and Mto1/2[bonsai] in the absence versus presence of Mzt1. Mzt is required for efficient copurification of Mto1[bonsai] with γ-TuSC. Note that the total yield of γ-TuSC proteins is also decreased. Western blots of protein inputs are shown in Figure S4A.
(B) Quantification of copurification of the indicated proteins from (A). Values for copurification are expressed as ratios, to correct for differences in yield, and normalized to values obtained in the presence of Mzt1.
(C) SDS-PAGE and anti-Mzt1 immunoblot of glutathione-agarose (GST) purification of coexpressed Mto1/2[bonsai]GST-Mto1[bonsai] and γ-TuSC in the absence versus presence of Mzt1. Mzt is required for efficient copurification of Alp6 with Mto1/2[bonsai]. Anti-Alp6 western blot indicates comparable Alp6 input levels in absence versus presence of Mzt1.
(D) Quantification of copurification of the indicated proteins in (C). Quantification was based on western blots shown in Figure S4B. Equivalent experiments using Mto1/2[bonsai]Strep-Mto1[bonsai] are shown in Figures S4C–S4E.
(E) SDS-PAGE of amylose (MBP) purification of Alp4-MBP or Alp6-MBP coexpressed with Mzt1. Mzt1 copurifies with Alp6-MBP but not with Alp4-MBP. Anti-Mzt1 western blot indicates comparable Mzt1 levels in the two inputs.
(F) SDS-PAGE and anti-Mzt1 western blot of glutathione-agarose purification of coexpressed Mto1/2[bonsai]GST-Mto1[bonsai] and Mzt1 in the absence versus presence of γ-TuSC. Mzt1 does not copurify with Mto1/2[bonsai] unless γ-TuSC is also present. The graph shows quantification of anti-Mzt1 western blot, normalized to value in first lane.
(G) Superose 6 size-exclusion chromatography of Alp6-MBP purified alone (blue) or from coexpression with Mzt1 (red). Alp6-MBP elutes in void volume, while Alp6-MBP:Mzt1 elutes much later. Inset shows SDS-PAGE of indicated fractions for Alp6-MBP:Mzt1.
(H) SDS-PAGE of amylose (MBP) purification of coexpressed Alp4-MBP and Mto1/2[bonsai] in the absence versus presence of Mzt1. Mto1/2[bonsai] copurifies with Alp4-MBP, independent of Mzt1 and other γ-TuSC proteins. The control experiment for non-specific binding of Mto1/2[bonsai] to amylose is shown in Figure S4H.
See also Figure S4.