Figure 5. Yeast two hybrid analysis of Bud14 and Bud14 mutants with spindle position checkpoint (SPOC) proteins.

(A) Bud14 interacts with Bfa1 but not with Bub2 or Kin4. (B) Bud14-Bfa1 interaction is dependent on Bub2. (C) Bfa1-Bud14 interaction is reduced in Bud14∆SH3 mutant. SGY37 was co-transformed with indicated plasmids. Empty plasmids served as a control for any self-activation. Kel1 served as a positive control. Cells were grown for 2 days on selective agar plates before overlay. Blue color formation was monitored as an indication of protein-protein interaction.

Figure 5—figure supplement 1. Analysis of Glc7 interaction with spindle position checkpoint (SPOC) components.

Yeast two hybrid assays of Glc7 with Bfa1, Bub2, and Kin4. Bud14 was included as a positive control.

Figure 5—figure supplement 2. SH3 domain of Bud14 is important for Bud14 function in spindle position checkpoint (SPOC).

(A) Endpoint analysis of SPOC deficiency index in bud14∆ kar9∆ cells carrying URA3-based empty plasmid (empty) or BUD14-containing URA3-based plasmids (BUD14, bud14-ΔSH3). Values for controls (empty and BUD14) are identical to those shown in Figure 3A as experiments were performed together. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. *p<0.05 according to Student’s t-test. (B) Serial dilutions of indicated strains bearing URA3-based empty plasmid (not indicated on figure) or BUD14-containing URA3-based plasmids (BUD14 and bud14-ΔSH3) were spotted on SC-URA plate and grown at indicated temperatures. Controls (lanes 1 and 2) are identical to those shown in Figure 3B as all drops come from the same agar plate.