n of the mitochondrial protein aconitase, a well-established method to monitor mitophagy, we confirmed that AG-221 chemical information rapamycin treatment in wild type cells exponentially growing in YPD medium induced mitophagy, since the protein was not detectable after 3 h of treatment with the compound. In contrast, when ptc6 mutant cells were exposed to the same concentration of rapamycin, Aco1 was still detectable for at least 6 h, indicating that rapamycin-induced mitophagy in wild type cells is, at least in part, dependent of Ptc6. This behavior is similar to that of cells lacking one of the main The rapamycin-induced transcriptional response is attenuated in ptc6 mutant cells Short-term treatment of wild type cells with rapamycin results in strong remodeling of gene expression. To identify the effect of ptc6 Functional Characterization of 22038495 Yeast Ptc6 mutation in the global expression pattern after rapamycin treatment, 
we performed microarray experiments comparing the rapamycin-induced transcriptional response of wild type and ptc6 mutants. Considering only the genes with valid data in both experiments we found that rapamycin caused changes in the expression levels of 1115 genes in wild type cells. In ptc6 mutant cells, rapamycin changed the levels of 884 transcripts, being the number of up-regulated and down-regulated genes of 375 and 509, respectively. In the case of the ptc1 ptc6 mutant, from the total number of genes with valid data, the rapamycin-induced genes were 417 and the down-regulated ones were 564 . These figures suggest that lack of Ptc6 may cause an attenuation of the transcriptional response to rapamycin. The alterations provoked by the ptc1 and ptc6 mutations in the response induced by rapamycin were verified in a set of four rapamycinresponsive, nitrogen catabolite repression regulated genes, required for adaptation to nonpreferred nitrogen sources and whose expression is controlled by the Gln3 transcription factor. As shown in Fig. 6B, when cells were challenged with rapamycin, it became evident that the response of these genes to the drug was attenuated in Ptc6-deficient cells, although somewhat less than in ptc1 cells. The transcription attenuation of the GAP1 and MEP1 genes in response to rapamycin in the ptc6 strain was also validated by RT-PCR. To explore the extent of the transcriptional attenuation in the response to rapamycin caused by the ptc6 mutation, we plotted the transcriptional changes for wild type and ptc6 after rapamycin treatment, and the value of the slope of the fitted line obtained by simple linear regression was taken as an ��attenuation index”. Therefore, if the global responses to the drug in both strains were similar, the expected slope value would be close to unity, whereas a weakened response in the ptc6 cells would decrease this index. The values of the slope for the genes consider up- ad down-regulated by rapamycin in ptc6 cells respect wild type cells were 0.8186 and 0.7424. 23713790 As a reference, the corresponding indexes for the same treatment with rapamycin in ptc1 respect wild type cells were 0.7502 and 0.9268, respectively. We also performed a similar calculation with the ptc1 ptc6 cells, and the corresponding slopes were 0.6671 and 0.8869. Thus, changes in the transcriptional profiles in response to rapamycin were attenuated in all three mutants analyzed, being this attenuation more relevant for the downregulated genes in the ptc6 strain, and for the up-regulated genes in the ptc1 ptc6 strain. One of the cell
