Figure 3.

Toxicity of Ugp1 overexpression is rescued by overexpression of PSK1 or PSK2. The PSK1psk2 (JRY277) strain overexpressing UGP1 (pJR3020b) displays a gal^ts growth defect that may be rescued by over-expression of PSK1 or PSK2. A trp plasmid overexpressing UGP1 from the TetO regulatable promoter was constructed for this screen (pJR3020b) using the system of Gari et al.⁵⁴ The JRY277 strain (PSK1psk2) was freshly transformed with either empty vector control (wild type, uppermost sample) or pJR3020b (remaining samples), and then one of five pRS426 constructs (empty vector, Psk1, Psk1 K1125R, Psk2, Psk2 K870R) and plated on SD-ura-trp + dox. The K1125R and K870R PAS kinase mutations (Km) are in the kinase domain and were constructed to decrease kinase activity.²³ Liquid cultures were then grown for 48 hours in the same media and were used for serial dilution into water 1:20, 1:5, 1:5, 1:5 and 1:5. Each dilution was then spotted (5 uL) onto an SGal-ura-trp plate and incubated at 30°C. A control plate was made on SD-ura-trp + dox and the lack of phenotype for any of the five strains was confirmed (all dilutions were comparable). The gal^ts phenotype of overexpressed UGP1 is not present in cells exposed to doxycycline. In contrast to the Psk2 Km mutation, the K1125R mutation in Psk1 appears to have little to no effect on its ability to suppress.