(A) The three-part screening strategy to identify Ypk1 substrates is shown schematically as a flow chart. Numbers indicate the number of hits/considered genes at each step in the screen. (B) The bioinformatic approach towards identifying Ypk1 substrates is schematized as a flowchart with each filter as a box. Genes were first filtered by MOTIPS on the basis of having likely phosphorylatable Ypk1 motifs. Subsequently, substrates were filtered by having many Ypk1 motifs or having a Ypk1 site known to be phosphorylated in published data sets. Lastly, genes were filtered by requiring the gene to have a published chemical sensitivity like Ypk1 does, or a published interaction with Ypk1, Ypk1 regulators (TORC2 or PP2A) or sphingolipid biosynthetic machinery. (C) A possible explanation for Ypk1 synthetic dosage lethality interactions is shown. Normally, the cell has enough kinase activity to buffer overexpression of a substrate (Substrate 2), so that essential substrates are regulated and normal growth is unperturbed. However, concurrent decrease in kinase activity coupled with substrate overexpression causes loss of regulation of essential substrate(s) (Substrate 1) leading to observable growth defects. (D) ypk1-as ypk2Δ (yAM135–A) cells were transformed with PGAL1-GFP (negative control), PGAL1-Orm1 or PGAL1-Orm2 (known Ypk1 substrates, positive SDL controls) plasmids. Overnight cultures were then serially diluted onto either dextrose (to repress substrate overexpression) or galatose (to induce substrate overexpression) containing media with increasing concentrations of the Ypk1-as inhibitor 3-MB-PP1. (E) GST-Orm1(1–85) (pFR203) and GST-Fps1(1–255) (pBT6) were purified from E. coli and incubated with [γ-32P]ATP and Ypk1-as, purified from S. cerevisiae, in the absence or presence of 3-MB-PP1. The products were then resolved by SDS/PAGE and analyzed as described in ‘Materials and methods’.