Surprisingly, we observed that pre-treatment of growing cultures of wild type cells with cycloheximide, a protein synthesis inhibitor, fully suppressed Pmk1 activation during glucose exhaustion (Figure 4B, upper panel). Moreover, this response appears to be specific since a strong Pmk1 activation
was observed in cycloheximide-treated and untreated cells under saline stress (Figure 4B, lower panel). These results strongly support that in fission yeast the stress by glucose limitation signals to the cell integrity pathway through a hitherto unknown mechanism which PD0332991 ic50 requires de novo protein synthesis. Figure 4 Pmk1 activation in response to glucose deprivation is independent on the SAPK pathway and requires de novo protein synthesis. A. Strains MI200 (Pmk1-Ha6H; Control), and MI204 (sty1Δ, Pmk1-Ha6H), were grown in YES medium plus 7% glucose to early-log phase and transferred to the same medium with 3% glycerol. Aliquots were harvested at timed intervals and Pmk1 was purified by affinity chromatography. Either activated or total Pmk1 were detected by immunoblotting with anti-phospho-p44/42 or anti-HA antibodies, respectively. B. Control strain MI200 (Pmk1-Ha6H)
was grown in YES medium plus 7% glucose to early-log phase, treated with of 100 μg/ml cycloheximide (CHX) for 60 min, and either transferred to the same medium with 3% glycerol (upper panel) or treated with 0.6 M KCl. Purification and detection of active or total Pmk1 was performed as described above. Pmk1 reinforces fission yeast LDN-193189 price adaptive response to metabolic stress imposed by glucose limitation To explore the biological significance of Pmk1 role during glucose deprivation we first determined whether the absence 4��8C of this MAPK might affect cell viability during growth adaptation from a glucose-based medium
to another with a non-fermentable carbon https://www.selleckchem.com/products/PD-173074.html source. In this context, it has been described that the SAPK pathway and its effector Sty1 are critical in fission yeast to allow adaptation from fermentative to respiratory metabolism [12, 13]. This is confirmed by results in Figure 5A, indicating that, contrary to wild type cells, the growth of sty1Δ cells was impaired when transferred from YES medium to a similar medium in which 7% glucose was substituted by 2% glycerol plus 3% ethanol. The shift to a medium containing 3% glycerol plus 0.05% glucose yielded the same results (not shown). Notably, either pmk1Δ cells or a mutant strain expressing a catalytically dead version of the MAPK Pmk1 displayed a growth defect in respiratory medium that was not observed in the presence of glucose (Figure 5A). This defect did not alleviate by the addition of NAC to the culture medium (Figure 5A), suggesting that endogenous oxidative stress was not the cause underlying this phenotype.