The Mkt1/Pbp1 complex negatively regulates TORC1 signaling during respiratory growth. (A) Growth curves of indicated strains grown in batch SL cultures plotted from OD₆₀₀ measurements. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells exhibited increased growth. P values were calculated with unpaired, two-sided t test (mean ± SD, n = 3). **P < 0.01. (B) GFP cleavage assay depicting rescue of autophagy following treatment with 200 nM rapamycin in pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells grown in SL medium for 6 h. (C) GO term analyses of TMT-MS experiment with pbp1∆ cells grown for 3 h in SL medium. Protein categories overrepresented among proteins with significantly increased abundances (fold-change > log₂(1.25)) are depicted. (D) GO term analyses of TMT-MS experiment with mkt1∆ cells grown for 3 h in SL medium. Protein categories overrepresented among proteins with significantly increased abundances (fold-change > log₂(1.25)) are depicted. (E) Venn diagram depicting number of proteins with significantly increased abundances identified in both mkt1∆ and pbp1∆ cells in the TMT-MS experiment. (F) Western blot depicting the phosphorylation of the TORC1 substrate Sch9 at Thr737 before and following switch from YPL to SL medium. Sch9 phosphorylation was monitored using an antibody specific for phospho-Thr737. All KO cells exhibited robust phospho-Thr737 signal at the 6 h time point. Cells with cycloheximide (25 μg/ml) and rapamycin (200 nM) served as positive and negative controls, respectively. (G) co-IP assessing interactions between Flag-tagged Mkt1 and Pbp1 with Kog1-HA 4 h following growth in either SD or SL media. Mkt1-Flag and Pbp1-Flag interacted preferentially in SL medium with Kog1-HA. Mkt1-Flag did not interact with Kog1-HA in pbp1∆ cells, while Pbp1-Flag interacted with Kog1-HA in mkt1∆ cells. Source data are available for this figure: SourceData F6.