FIG. 6.

Effects of mutant PML proteins on self-interaction measured with a yeast two-hybrid assay. (Left) Diagram illustrating the structure of the GAL4-A/PML fusion proteins used. The two major translocation fusion points occurring within the PML protein (at positions 552 and 955) in PML/RARα fusions in APL are indicated by arrows. The proposed NLS (at positions 467 to 490) is indicated (36). The location of the paired RING finger point mutations (C₈₈P₈₉→S₈₈R₈₉) is indicated by a star. The amino acid positions of the restriction enzyme cleavage sites used to generate the GAL4-A/mutant PML fusion are indicated. Dotted bars, N-terminal Pro-rich domain and C-terminal Ser-rich domain; black bars, RING finger domain (left) and two adjacent Cys/His-rich domains (right); hatched bars, putative α-helical region. Av, AvrII; Pv, PvuII; Sm, SmaI. (Right) Qualitative and quantitative results of the yeast self-interaction assay. No detectable β-galactosidase activity was measured in Y190 cells transformed with the plasmid encoding the GAL4-DB/PML(1-560) fusion protein alone. Plasmids encoding a variety of GAL4-A/PML fusion proteins were then introduced together with GAL4-DB/PML(1-560) into Y190 cells. Transformants were selected on plates lacking Trp and Leu, and β-galactosidase activity of the transformants was measured as described in Materials and Methods. Lines: 1, GAL4-A/PML(1-560) in pJHA266; 2, GAL4-A/PML(1-560, C₈₈P₈₉→S₈₈R₈₉) (pJHA267); 3, GAL4-A/PML(1-447) (pJHA268); 4, GAL4-A/PML(1-267) (pJHA269); 5, GAL4-A/PML(1-96) (pJHA270); 6, GAL4-A/PML(224-560) (pJHA281); 7, GAL4-A/PML(447-560) (pJHA271); 8, GAL4-A/PML(1-560, Δ281-304) (pEB5); 9, GAL4-A/PML(1-560, C₈₈P₈₉→S₈₈R₈₉ Δ281-304) (pEB6). ^aMean values for β-galactosidase units for interaction between GAL4-DB/PML(1-560) and GAL4-A/PML(1-560) in duplicated assays are indicated as 100%. The relative activity of control SNF1/SNF4 interaction in the same assay was 148%. ^bSelf-interaction with the GAL4-DB/PML(96-560, Δ281-304) fusion protein encoded by plasmid pJHA294.