The genetic interactions between SKN7 and MBP1. (A) mbp1 mutations confer resistance to pGAL-SKN7–dependent lethality. Derivatives of the wild-type strain W303 (WT) were transformed with either the pMW20 empty vector (pGAL) or the pGAL-SKN7 construct. Transformants were streaked on galactose-containing minimal agar plates and incubated at 30°C. (B) skn7 mutations confer resistance to pGAL-MBP1–dependent lethality. Derivatives of the wild-type strain W303 were transformed with either the pEMBLyex4 empty vector (pGAL) or the pGAL-MBP1 construct. Transformants were streaked on minimal agar plates containing galactose and lacking leucine and incubated at 30°C.

Overexpression of MBP1 or SKN7 arrests division of cells. W303-1A cells containing pGAL-SKN7 or pGAL-MBP1 were grown at 30°C in YNB medium containing raffinose. Galactose was added, and incubation continued for the equivalent of two generations. (A) Cellular morphology (upper panels) and DAP1 staining (lower panels). (B) FACS analysis. Note that DNA peaks tend to migrate to the right as cells enlarge during cell cycle arrest.

SKN7 function is required for MBP1 suppression of the swi4^ts swi6Δ mutant. (A) Skn7 is necessary for suppression of swi4^ts swi6Δ by high-copy-number MBP1. The swi4^ts swi6Δ and swi4^ts swi6Δ skn7Δ strains were transformed with YEp24 or a multicopy plasmid carrying either SWI4 or MBP1 and streaked on YEPD agar plates at 37°C. (B) Skn7 is required for increased cyclin levels in Mbp1 suppression of a swi4^ts swi6Δ strain. The appropriate strains (see text) were grown to midlog phase in YNB glucose at 25°C, transferred to 37°C for the times shown, and sampled, and total RNA was extracted and a Northern blot was prepared (). Probes were internal fragments of the genes concerned, and RPB4 was used as a loading control. This experiment was repeated, and an average of the data points is presented above with error bars, but raw data are shown for only one experiment. For the two sets of data to be directly comparable, levels are presented relative to the level at 0 time of strain K2003 containing vector alone. This value was arbitrarily set at 1. Note that where no error bar is shown, the difference between the two samples concerned was insignificant and the graphing package used was unable to draw the error bars. Quantitation was by phosphorimager.

Skn7 is not part of the core MBF. MBF was analyzed by gel retardation with a radioactively labeled 3xMCB probe (). (A) Deletion of SKN7 does not affect MBF. Increasing amounts of crude extract were assayed from the strains shown. Note that the mbp1 mutation used is a point mutation (an allele of bor2) rather than a deletion. (B) Antibodies to Skn7 do not supershift MBF. Lane 1, free probe; lanes 2–10, wild-type (W303) crude extract; lane 2, no competitor; lane 3, competition with a 100-fold excess of the cold probe; lanes 4–6, preimmune serum (1:100, 1:200, 1:500); lanes 7–9, anti-Skn7 antibodies (1:100, 1:200, 1:500); lane 10, anti-Swi6 antibodies (1:200); lane 11, W303 skn7Δ crude extracts with anti-Swi6 antibodies (1:200).

In vitro interaction between Skn7 and Mbp1. (A) The interaction between Skn7 and Mbp1 in GST-pulldown experiments. The fusion proteins GST-Skn7 (lanes 7–10) and GST-Swi6 (lanes 11–13) and the negative control GST alone (lanes 5 and 6) were purified on glutathione beads and incubated with in vitro translated, radioactively labeled Mbp1 (lanes 5, 7, and 11), Skn7 (lanes 6, 8, and 12), Swi4 (lanes 9 and 13), or Swi6 (lane 10). The bound proteins were separated by SDS-PAGE and visualized by autoradiography. The total amount of radioactive protein added to each binding reaction is shown in lanes 1 (Mbp1), 2 (Skn7), 3 (Swi4), and 4 (Swi6). (B) Schematic representation of the mutant derivatives of GST-Skn7 used to identify the regions of interaction of Skn7 with Mbp1. The different domains of Skn7 are illustrated, and the amino acid positions of the truncations and internal deletions are indicated. DBD, DNA-binding domain; pAB81, skn7ΔHR (see text). (C) The different derivatives of the GST-Skn7 fusion protein were assayed for their ability to bind in vitro translated, radioactively labeled Mbp1 in GST-pulldown experiments. The total amount of radioactive Mbp1 added to each binding reaction is shown (lane L). GST (lane 7) was used as a negative control.

Genetic interactions between SKN7, MBP1, and genes involved in bud emergence. (A) pGAL-SKN7–dependent lethality is suppressed by high-copy-number CLN1. W303-1A containing pAB56 (2-μm, TRP1, GAL-SKN7) was transformed with YEp24 or a multicopy plasmid carrying CLN1, CLB5, or CLB6. Transformants were streaked on galactose-containing minimal agar plates and incubated at 30°C. (B) The temperature sensitivity of a cdc42-1 mutant is enhanced by high-copy-number SKN7. A cdc42-1 strain was transformed with the plasmids shown and streaked onto YEPD agar plates at 34°C. (C) pGAL-CDC42A118–dependent lethality is suppressed by skn7 and mbp1 mutations. The pGAL-CDC42A118 plasmid was transformed into W303, W303 swi6Δ, W303 skn7Δ, and W303 mbp1/bor2. The transformants were streaked onto galactose-containing minimal plates at 30°C.