The Mkt1/Pbp1 complex is required for autophagy during respiratory growth. (A) Images of WT, pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells before and 6 h after switching from YPL to SL medium. Localization of GFP to the vacuole (Vph1-mCherry) indicates mitophagy (Kanki and Klionsky, 2008). pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells were unable to induce mitophagy following switch to SL medium. Scale bar = 1 μm. (B) GFP cleavage assay showing mitophagy in indicated strains following switch from YPL to either SL or SD-N (high glucose, nitrogen starvation) media for 6 h. The accumulation of free GFP indicates mitophagy. Mitophagy was defective in pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells in SL medium. (C) GFP cleavage assay showing pexophagy in indicated strains following switch from YPL to either SL or SD-N media for 6 h. Pexophagy was defective in pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells in SL medium. (D) Alkaline phosphatase assay assessing general autophagy in cells 7 h following switch from YPL to SL medium. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells had reduced alkaline phosphatase activity reflective of a general autophagy defect. P values were calculated using unpaired, two-sided t test (mean ± SD; n = 4). ***P < 0.001. (E) Alkaline phosphatase assay measuring mitophagy in cells 7 h following switch from YPL to SL medium. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells had reduced alkaline phosphatase activity indicative of a mitophagy defect. P values were calculated using unpaired, two-sided t test (mean ± SD; n = 4). **P < 0.01; ***P < 0.001. (F) Pbp1 overexpression rescues mitophagy in mkt1∆ cells following switch from YPL to SL medium for 6 h. Pbp1 was overexpressed by swapping the PBP1 promoter for the TEF1 promoter. Mitophagy was assayed using the Idh1-GFP cleavage assay. (G) GFP cleavage assay showing Pbp1Δ500-503 and Pbp1-4A strains had reduced Pbp1 protein levels and disrupted mitophagy following growth in SL medium for 6 h. Note: residual free GFP in Pbp1Δ500-503 and Pbp1-4A cells was detected, which may reflect continued TORC1 repression. Source data are available for this figure: SourceData F5.
Figure 5.

The Mkt1/Pbp1 complex is required for autophagy during respiratory growth. (A) Images of WT, pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells before and 6 h after switching from YPL to SL medium. Localization of GFP to the vacuole (Vph1-mCherry) indicates mitophagy (Kanki and Klionsky, 2008). pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells were unable to induce mitophagy following switch to SL medium. Scale bar = 1 μm. (B) GFP cleavage assay showing mitophagy in indicated strains following switch from YPL to either SL or SD-N (high glucose, nitrogen starvation) media for 6 h. The accumulation of free GFP indicates mitophagy. Mitophagy was defective in pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells in SL medium. (C) GFP cleavage assay showing pexophagy in indicated strains following switch from YPL to either SL or SD-N media for 6 h. Pexophagy was defective in pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells in SL medium. (D) Alkaline phosphatase assay assessing general autophagy in cells 7 h following switch from YPL to SL medium. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells had reduced alkaline phosphatase activity reflective of a general autophagy defect. P values were calculated using unpaired, two-sided t test (mean ± SD; n = 4). ***P < 0.001. (E) Alkaline phosphatase assay measuring mitophagy in cells 7 h following switch from YPL to SL medium. pbp1∆, mkt1∆, and mkt1∆pbp1∆ cells had reduced alkaline phosphatase activity indicative of a mitophagy defect. P values were calculated using unpaired, two-sided t test (mean ± SD; n = 4). **P < 0.01; ***P < 0.001. (F) Pbp1 overexpression rescues mitophagy in mkt1∆ cells following switch from YPL to SL medium for 6 h. Pbp1 was overexpressed by swapping the PBP1 promoter for the TEF1 promoter. Mitophagy was assayed using the Idh1-GFP cleavage assay. (G) GFP cleavage assay showing Pbp1Δ500-503 and Pbp1-4A strains had reduced Pbp1 protein levels and disrupted mitophagy following growth in SL medium for 6 h. Note: residual free GFP in Pbp1Δ500-503 and Pbp1-4A cells was detected, which may reflect continued TORC1 repression. Source data are available for this figure: SourceData F5.

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