Model of the molecular regulation of Cdc48p nuclear targeting by tyrosine phosphorylation. Affinity (green arrows) of the amino-terminal and the carboxy-terminal elements with the central region bring nuclear targeting signal and acidic domain in proximity, masking the signal. Tyrosine phosphorylation abolishes the attachment, resulting in a conformational change and an exposition of the targeting signal.

Human VCP accumulates at the centrosomes during mitosis. Cultivated WISH cells in interphase (A), metaphase (B and C), or anaphase (D). Cell shape is shown by phase-contrast microscopy (pc). VCP is visualized by immunofluorescence microscopy using FITC-labeled secondary antibodies, and phosphotyrosine (Tyr-P) using rhodamine-labeled secondary antibodies. DNA is stained with bisbenzimide. A cell–cell contact staining with anti-phosphotyrosine antibody is marked by an arrow. Scale bar, 10 μm.

Cdc48p nuclear import requires an amino-terminal nuclear localization sequence and the phosphorylation of a carboxy-terminal tyrosine residue. Mutant forms of Cdc48p were visualized by immunofluorescence microscopy using anti-Cdc48p antiserum and FITC-labeled secondary antibody; DNA was stained with 4,6-diamidino-2-phenylindole. The strains were grown on YEP containing 4% glucose and expressed Cdc48p^nonuc (A), Cdc48p^Y834F (B), Cdc48p^Y834E (C), or Cdc48p^Y834F/noacid (D) under the control of a GAL1 promoter. Scale bar, 10 μm.

Phosphotyrosinylation of VCP is confined to mitosis. (A) WISH cells synchronized by thymidine treatment. Culture 9 h after release from thymidine block (panel 1) and exponentially growing untreated control culture (panel 2) stained with crystal violet. Scale bar, 50 μm. (B) Total extracts of WISH cells harvested at t = 4 h (lanes 1 and 7), t = 8 h (lanes 2 and 8), t = 9 h (lanes 3 and 9), t = 10 h (lanes 4 and 10), t = 11 h (lanes 5 and 11), t = 12 h (lanes 6 and 12) after release from the thymidine block hybridized with anti-VCP antibody (lanes 1–6) or with anti-phosphotyrosine antibody (lanes 7–12), respectively. Fifty micrograms of total protein were applied in each lane.

Cdc48p is tyrosine phosphorylated in growing cells. (A) Exponentially grown wild-type strain (extract treated with 8 mU phosphotyrosine phosphatase for 3 h [lane 2], untreated extract [lane 3]), exponentially grown cdc48^Y834F strain (lane 4), exponentially grown cdc48^Y834E strain (lane 5), stationary wild-type strain (lane 6) hybridized with anti-phosphotyrosine antibody; control: wild-type strain hybridized with anti-Cdc48p antibody (lane 1). Cells were grown on YEP containing 4% galactose. (B) Flu epitope-tagged Cdc48p (lanes 1 and 4) and wild-type Cdc48p (lanes 2 and 3) hybridized with anti-Cdc48p antibody (lanes 1 and 2) or anti-phosphotyrosine antibody (3, 4). (C) Synchronized cdc20 cells at t = 45 min (lanes 1 and 5), t = 60 min (lanes 2 and 6), t = 75 min (lanes 3 and 7), t = 90 min (lanes 4 and 8) after reshift to the permissive temperature hybridized with anti-phosphotyrosine antibody (lanes 1–4) or with antiCdc48p antibody (lanes 5–8), respectively. Western blots of total cell extracts. Fifty micrograms of total protein were applied in each lane.

Cdc48p is localized in the nucleus in late G₁, and is excluded from the nucleus in the other phases of the cell division cycle. Cdc48p was visualized by immunofluorescence microscopy using fluorescein isothiocyanate (FITC)- or rhodamine-labeled secondary antibody, and DNA was stained with 4,6-diamidino-2-phenylindole. Exponentially grown S. cerevisiae wild-type cells (A) synchronized cdc20^ts mutants at t = 60 min (B) and t = 75 min (C) after reshift to the permissive temperature; cdc1^ts mutant arrested at 37°C for 3 h (D) and 2 h after reshift (E) to the permissive temperature. Scale bar, 10 μm. Distribution of cell morphology of a cdc20 culture reshifted to 28°C after 3 h incubation at 37°C (F). □, portion of unbudded cells; ◊, portion of tiny budded cells; ○, portion of small budded cells; ▵, portion of large budded cells. Nuclear (lanes 1–4) and microsomal (lanes 5–8) fractions of synchronized cdc20 cultures harvested at t = 0 min (lanes 1 and 5), t = 15 min (lanes 2 and 6), t = 30 min (lanes 3 and 7), and t = 45 min (lanes 4 and 8) after shift to the permissive temperature hybridized with anti-Cdc48p-antibody (G). Fifty micrograms of total protein were applied in each lane.