Sea3 functions through the TORC1 pathway in response to DNA damage
Given Sea3′s role as a negative regulator of the Iml1 complex (SEACIT), which in turn negatively regulates TORC1 (Panchaud et al. 2013a,b), we reasoned that the delay in colony formation in the sea3∆ BIR assay strain might be the result of hyperrepression of TORC1 (Figure 4A). If so, then deletion of IML1 would rescue the delay. This is precisely what we observed (Figure 4B). Similarly, we found that deletion of IML1 rescued the sea3Δ mutant growth delay on bleomycin in the YPH274 strain background (Figure S4). Thus, these data are consistent with Sea3 functioning through TORC1 and this regulation of TORC1 impacting colony formation post-DNA repair in the BIR assay strain.
Figure 4  Sea3 functions through TORC1, but not autophagy, in response to DNA damage. (A) Proposed effects on TORC1 if Sea3 functions as a negative regulator of the Iml1 complex/SEACIT. (B) Fivefold serial dilutions of wild-type and iml1Δ, sea3Δ iml1Δ, and sea3Δ BIR assay strain mutants plated on YPD and YPGal. (C) Fivefold serial dilutions of wild-type and atg5Δ, sea3Δ atg5Δ, and sea3Δ BIR assay strain mutants plated on YPD and YPGal. To determine whether Sea3 and, perhaps, TORC1 signaling were required for growth in response to other stress conditions, we plated sea3∆ mutants in both the YPH274 and BIR assay strain backgrounds on YPD containing a low concentration of glucose (0.25% compared to 2% in standard YPD), high salt (0.5 M NaCl added to standard YPD), or hydrogen peroxide (3 mM H2O2 added to standard YPD), and at high temperature (37°). We found the sea3Δ mutant in the YPH274 strain background had a growth delay on medium containing high salt, an effect that was mediated through TORC1 signaling as it was rescued by deletion of IML1 (Figure S5A). However, a growth delay was not observed on high salt with the sea3∆ mutant in the BIR assay strain background (Figure S5B), suggesting the phenotype was influenced by strain specific factors and not solely the absence of Sea3. In both strain backgrounds, we found that Sea3 was not required for growth in response to any of the other stresses tested (Figure S5). Taken together, although there are some strain specific differences, sea3Δ mutants experience a growth defect under conditions that induce DSBs and under conditions of high salt in a TORC1-dependent manner.
We next looked for possible targets downstream of TORC1 that might be responsible for the delay in colony formation phenotype. Likely candidates were factors mediating autophagy, which is negatively regulated by TORC1 signaling, and previously identified to be a pathway downstream of the yeast SEA complex (Dokudovskaya et al. 2011; Takahara and Maeda 2013). If Sea3 functioned to promote TORC1 repression of autophagy and, thereby, regulate growth post-DNA repair, then a block in autophagy would rescue the delay observed in the sea3∆ mutant. However, we found that deletion of ATG5, which encodes a core autophagy factor (Mizushima et al. 1998), had no impact on the delay in the BIR assay strain (Figure 4C). Thus, the delay in colony formation in the sea3∆ mutant was not due to aberrant up-regulation of autophagy but rather due to misregulation of another downstream TORC1 target.