Distinct Subsets of Sit4 Holophosphatases Are Required for Inhibition ofSaccharomyces cerevisiaeGrowth by Rapamycin and Zymocin

Abstract

Protein phosphatase Sit4 is required for growth inhibition of Saccharomyces cerevisiae by the antifungals rapamycin and zymocin. Here, we show that the rapamycin effector Tap42, which interacts with Sit4, is dispensable for zymocin action. Although Tap42 binding-deficient sit4 mutants are resistant to zymocin, these mutations also block interaction between Sit4 and the Sit4-associating proteins Sap185 and Sap190, previously shown to mediate zymocin toxicity. Among the four different SAP genes, we found that SAP190 deletions specifically induce rapamycin resistance but that this phenotype is reversed in the additional absence of SAP155. Similarly, the rapamycin resistance of an rrd1⌬ mutant lacking the Sit4 interactor Rrd1 specifically requires the Sit4/Sap190 complex. Thus, Sit4/Sap190 and Sit4/Sap155 holophosphatases apparently play opposing roles following rapamycin treatment, although rapamycin inhibition is operational in the absence of all Sap family members or Sit4. We further identified a Sit4-interacting region on Sap185 in sap190⌬ cells that mediates Sit4/Sap185 complex formation and is essential for dephosphorylation of Elp1, a subunit of the Elongator complex. This suggests that Sit4/Sap185 and Sit4/Sap190 holophosphatases promote Elongator functions, a notion supported by data showing that their inactivation eliminates Elongator-dependent processes, including tRNA suppression by SUP4 and tRNA cleavage by zymocin.

A Novel Sit4 Phosphatase Complex Is Involved in the Response to Ceramide Stress in Yeast

“…However, mutation of the four SAP regulatory proteins, either individually or in combination, does not confer resistance to dihydroceramide (Figure 2(a)). In previous studies, the phenotype of the quadruple sap mutant has been indistinguishable from that of sit4 Δ [19, 20]. Thus, the ceramide sensitivity of the sap mutant is the first observed separation of function between sit4 Δ and sap ΔΔΔΔ.…”

“…Diverse regulatory subunits of Sit4p are partially responsible for the different specificities of Sit4p; for example, Tap42p is phosphorylated by Tor and binds Sit4p [17] and Sap185p and Sap190p subunits are essential for correct phosphoregulation of Elongator and tRNA modification [18]. Mutation of the SAPs (Sit4 associated proteins) can confer different specificities on Sit4p but a deletion of all four SAPs always results in the same phenotypes as deletion of SIT4 , for example resistance to the tRNAse toxin zymocin, cell cycle arrest, and sensitivity to rapamycin [19, 20]. The accessory protein Kti12p is also essential for the phosphoregulation of the Elongator subunit Elp1p by the Sit4p/Sap185p/Sap190p complex.…”

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

“…Intriguingly, reintroduction of SAP190 into rrd1 ∆ sap ∆∆∆∆ cells reestablishes the drug resistance typical of rrd1 ∆ cells alone suggesting the trait depends on Sit4 and Sap190 17. This is similar to Sit4 dependent Elongator dephosphorylation, which requires Sap185 and Sap190 and promotes Elongator’s tRNA modification function 252728.…”

Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…Both lacked adenine to check for ade2-1 ochre read-through by the tRNA suppressor SUP4 and formation of adenine prototrophic cells (Jablonowski et al, 2006). The latter involved growth for 3 d at 30°C of 10-fold serial dilutions of yeast strains W303-1A and pGAL-GRX4/grx3D transformed with SUP4 on a single-copy plasmid pTC3 (Shaw and Olson, 1984;Jablonowski et al, 2009).…”