The Mkt1-G30D polymorphism underlies the capacity of yeast to undergo autophagy during respiratory growth. (A) Western blot showing CEN.PK and S288C cells expressing endogenously substituted Mkt1-D30 alleles had reduced Mkt1-Flag and Pbp1-HA protein levels 3 h following switch from YPL to SL medium. (B) co-IP experiments testing interactions between Mkt1-Flag and Pbp1-HA in CEN.PK and S288C cells 3 h following switch from YPL to SL medium. Mkt1-Flag interacted poorly with Pbp1-HA in CEN.PK and S288C cells expressing Mkt1-D30 alleles. (C) GFP cleavage assay depicting impaired mitophagy and reduced Mkt1 protein levels in CEN.PK Mkt1-D30 cells 6 h following switch from YPL to SL medium. (D) Growth curves of CEN.PK strains incubated in batch SL cultures plotted from OD₆₀₀ measurements. CEN.PK Mkt1-D30 cells phenocopied hyperproliferative growth of mkt1∆ cells. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 4). ***P < 0.001. (E) GFP cleavage assay reflecting endogenous substitution of Mkt1-G30 alleles rescued mitophagy and Mkt1-Flag protein levels in S288C yeast. Cells were collected before and 6 h following switch from YPL to SL medium. (F) Alkaline phosphatase assay to monitor general autophagy 6 h following growth in SL medium. S288C WT cells had decreased general autophagy, which was rescued by an endogenously substituted Mkt1-G30 allele. P values were calculated using unpaired, two-sided t test (mean ± SD, n = 4). **P < 0.01; ***P < 0.001. (G) GFP cleavage assay representing partially rescued mitophagy in S288C WT and Mkt1-D30 cells treated with rapamycin following 6 h in SL medium. (H) GFP cleavage assay showing Pbp1 overexpression partially rescued mitophagy defect in S288C WT cells grown in SL medium for 6 h. Source data are available for this figure: SourceData F9.