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. 2009 Sep 1;8(11):1637–1647. doi: 10.1128/EC.00205-09

FIG. 5.

FIG. 5.

Properties of Tap42 binding-deficient sit4 mutants. (A) Alignment of the Tap42 binding sites (boxed) of Sit4 and Sit4 homologs (human, PP6; Drosophila, PPV) (52). Substitutions of invariant residues in the different sit4 alleles are shown in boldface. (B) Killer eclipse assays and phosphomodification of Elongator subunit Elp1. ELP1-HA sit4Δ cells carrying wild-type SIT4 or sit4 alleles with the indicated single, double, and triple mutations in the Tap42 binding motif of Sit4 (52) were tested for zymocin inhibition (top) using the killer eclipse assay (33). Zymocin resistance (R) was distinguished from sensitivity (S). Total protein from these ELP1-HA expressers was extracted and immunoblotted using the anti-HA antibody (middle) to distinguish phosphorylated (circled P) Elp1 from unmodified Elp1 (27). Additional Western blots with anti-Pfk1 antibodies (bottom) detected α and β subunits of phosphofructokinase, which served as protein-loading controls. (C) Sit4/Sap155 and Sit4/Sap190 interaction studies. Plasmid-borne alleles of SAP155-HA and SAP190-HA were transformed into the indicated Tap42 binding-deficient sit4 mutants (52) and SIT4 wild-type cells. Following immune precipitation (IP) by anti-HA antibodies, the Sit4 and Sap proteins were detected in these precipitates (upper two blots) using anti-Sit4 and anti-HA antibodies and compared to the protein levels present in the input (preIP) controls (lower two blots). The arrows indicate the positions of Sap155, Sap190, and Sit4. (D) Rapamycin phenotypes. The indicated S. cerevisiae strains (wt, wild type) were assayed for sensitivity to 50 nM rapamycin (+ rap) using the tap42-11 mutant as a rapamycin-resistant control. Growth was for 3 to 4 days at 25°C. Rapamycin responses included resistance (R), sensitivity (S), and hypersensitivity (HS). Note that the Tap42 binding-deficient sit4 mutant (L35A E37A E38A) abolished dosage suppression of rapamycin by SAP155. (E) Heat-inducible zymocin resistance of the sit4-102 mutant, which has a Tap42 binding defect at 37°C. Shown are plate assays of the indicated tester strain dilutions at 30°C (top) and 37°C (bottom) on medium lacking (− zymo) or containing (+ zymo) 45% (vol/vol) zymocin. Sensitivity (S) and resistance (R) to zymocin are indicated. The sit4-102 allele was tested in SSD1-v (sit4-102/v) and ssd1-d (sit4-102/d) backgrounds. Thermosensitivity at 37°C is indicated (ts).