Yme1, Mgr1, and Mgr3 are essential for the degradation of Tom22 and Om45. (A) Topology of Tom22 and Om45. Cyt, cytosol. (B) The TOM22-HA, OM45-HA, and MDM34-HA strains in WT, cdc48-3, or cdc48^T413R background were grown in lactate media (YPL) to log phase at 25°C and then treated with CHX at 37°C (restrictive temperature). Anti-Por1 blots are shown as loading controls. cdc48-3 and cdc48^T413R are two temperature-sensitive (ts) mutations of Cdc48. (C) The TOM22-HA, OM45-HA, and FZO1-HA strains in WT, pre1^ts pre2^ts, or cim3-1 background were similarly analyzed as in B. pre1^ts pre2^ts and cim3-1 are ts mutations of the proteasome subunits Pre1, Pre2, and Cim3, respectively. (D–F) The WT and indicated mutant strains expressing either Tom22-HA or Om45-HA were grown in the indicated conditions to log phase and then treated with CHX and collected at the indicated time points. Molecular masses are shown in kilodaltons.