Figure 2.

Hrr25p localization and generation of a conditional HRR25. (A) GFP-Hrr25p is diffusely localized throughout the cell and concentrates at the bud tip and at the bud neck. YPH499 cells expressing GFP-Hrr25p (pKK191) were grown to log phase and examined (see Materials and Methods). (B) Schematic of the HRR25^degron allele expressed under a galactoseinducible promoter (pKK204). The N-terminal ubiquitin is cleaved cotranslationally, revealing the destabilizing arginine (the N-degron). The 23 amino acids of LacI provide an internal lysine residue to which the polyubiquitin chain is attached. Hrr25p^degron is then degraded by the proteosome. (C) α-HA Western blot showing the disappearance of Hrr25p^degron 5 h after addition of glucose (KKY387). (D) Abnormal cell morphology characteristic of hrr25 mutant cells seen 5 h after the addition of glucose to KKY387. Cells displaying this abnormal morphology represent 5% of all cells.