Figure 5.

Degradation of a Deg1-Vma12 membrane protein requires both Doa10 and Cdc48. (A) Schematic of the predicted topology of Deg1-Vma12-PrA (Deg1-VP). (B) Deg1-Vma12-GFP localizes to the ER. The PrA moiety in Deg1-VP was replaced with GFP to allow fluorescence localization in live cells (doa10Δ). Scale bar, 5 μm. (C) Deg1*-VP is an integral membrane protein based on cell fractionation. The Deg1 degron was inactivated by mutation (F18S, I22T: Deg1^*) to allow it to accumulate. (D) Deg1-VP is a short-lived substrate of the Doa10 pathway as measured by pulse-chase analysis. Proteins were precipitated with antibodies to α2. (E) Inactivation of the Deg1 degron (Deg1^*) blocks Deg1*-VP degradation. Analysis was performed as in (D). (F) Degradation of Deg1-VP requires the Cdc48 complex. Analysis was carried out as in (D), except that cells grown overnight at 24°C were shifted to 30°C for 4 h, and then to 37°C for 1.5 h prior to pulse-chase analysis at 37°C; quantitation is shown on the right.