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. Author manuscript; available in PMC: 2024 Jun 15.
Published in final edited form as: Mol Cell. 2023 May 15;83(12):2045–2058.e9. doi: 10.1016/j.molcel.2023.04.022

Figure 1. The mitochondrial protein Atg44 is essential for mitophagy.

Figure 1.

(A) The indicated S. pombe strains expressing Tuf1-RFP were grown in EMM and shifted to nitrogen-starvation medium (EMM-N). Cells were collected at the indicated time points and Tuf1-RFP processing was monitored by immunoblotting.

(B) Mitophagy was examined for the wild-type strain and the atg44Δ mutant exogenously expressing Sp-atg44+ or Sc-ATG44 by the Tuf1-RFP processing assay as in (A).

(C) The indicated S. cerevisiae strains expressing Idh1-GFP were grown in YPL and shifted to nitrogen-starvation medium (SD-N). Cells were collected at the indicated time points and Idh1-GFP processing was monitored by immunoblotting.

(D) Mitophagy in S. cerevisiae atg44Δ cells exogenously expressing Sc-ATG44, Sp-atg44+, or Kp-ATG44 was monitored using the Idh1-GFP processing assay as shown in (C).

(E) The indicated S. cerevisiae strains expressing Pho8Δ60 were grown in YPD and shifted to SD-N for 4 h. Samples were collected, and the protein extracts were assayed for Pho8Δ60 activity. The results represent the mean and standard deviation (SD) of four experiments.

(F) S. cerevisiae cells expressing Sc-Atg44-FLAG and Idh1-GFP were cultured in SMD and analyzed by immunofluorescence microscopy using an anti-FLAG antibody. Scale bar, 5 μm.

(G) Subcellular fractionation was conducted using S. cerevisiae. The total cell homogenate (Total cell) was fractionated by centrifugation to obtain a post-nuclear supernatant (PNS), a mitochondria-enriched pellet (Mito) and a supernatant (Sup). Atg33 and Pgk1 were detected as mitochondrial and cytosolic markers, respectively.

Histone H3 (A and B) and Pgk1 (C and D) were used as loading controls. The ratio of RFP to total RFP (Tuf1-RFP and RFP) (A and B) or GFP to total GFP (Idh1-GFP and GFP) (C and D) were quantified. The value of the wild-type strain at the 24-h time point (A and B), wild-type strain at the 6-h time point (C), or Sc-ATG44-expressing strain at the 6-h time point (D) was set to 100%. The results represent the mean and SD of three (A, B, and D) or four (C) experiments.

See also Figures S1 and S2.