1. Cell Biology
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Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast

  1. Tomoyuki Fukuda  Is a corresponding author
  2. Yuki Ebi
  3. Tetsu Saigusa
  4. Kentaro Furukawa
  5. Shun-ichi Yamashita
  6. Keiichi Inoue
  7. Daiki Kobayashi
  8. Yutaka Yoshida
  9. Tomotake Kanki  Is a corresponding author
  1. Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Japan
  2. Omics Unit, Niigata University Graduate School of Medical and Dental Sciences, Japan
  3. Department of Structural Pathology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Japan
Research Article
Cite this article as: eLife 2020;9:e61245 doi: 10.7554/eLife.61245
7 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Atg43 is required for starvation-induced mitochondrial degradation in S.?pombe.

(A, D) The indicated strains expressing Tuf1-mRFP were grown in EMM and shifted to the same medium without a nitrogen source (EMM–N). Cells were collected at the indicated time points after nitrogen starvation was initiated. Tuf1-mRFP processing was monitored by immunoblotting. Lower panel, long exposure (LE). (B) The indicated strains expressing Sdh2-GFP were collected at the indicated time points after shifting to nitrogen starvation medium. The processing of Sdh2-GFP was monitored by immunoblotting. (C) Schematic representation of the atg43-1 allele. The protein-coding region is depicted by a gray arrow. The nucleotide region that is deleted by the replacement with the kanMX4 gene (black arrow) is shown. (E, G) The indicated strains were grown in EMM, and their serial dilutions were spotted onto solid YES medium for the growth assay. (F) The protein-coding region of the atg43+ gene was cloned into an integration vector to express atg43+ under the control of the moderate adh31 promoter. The atg43-1 mutants integrated with the atg43+ gene (atg43+) or an empty vector (vector), as well as a wild-type strain, were collected at the indicated time points after induction of nitrogen starvation and were subsequently subjected to immunoblotting. (H–J) The indicated strains expressing GFP-Atg8, Pgk1-GFP, or Yop1-GFP were collected at the indicated time points after induction of nitrogen starvation. Degradation of each substrate was monitored by immunoblotting. Histone H3 was used as a loading control (LC) for immunoblotting.

Figure 1—figure supplement 1
Mitochondrial proteins are degraded by autophagy dependently on Atg43 and Tom70.

(A–D) The processing of tagged mitochondrial proteins depends on mitochondrial degradation by autophagy. Wild-type and the indicated mutant cells, which express Tuf1-mRFP (A and B), Tom70-GFP (C), or Mic60-GFP (D), were grown in EMM and shifted to EMM–N for nitrogen starvation. Cells were collected at the indicated time points after induction of nitrogen starvation, followed by immunoblotting. Histone H3 was used as a loading control (LC). (E) Autophagy does not affect vegetative growth. The indicated strains were grown in EMM, and their serial dilutions were spotted onto solid YES medium for the growth assay. (F–I) Atg43 and Tom70 are mitophagy-specific proteins. Wild-type and the indicated mutant cells expressing Tdh1-mRFP (F), GFP-Atg8 (G), Pgk1-GFP (H), or Yop1-GFP (I) were grown in EMM and shifted to EMM–N for nitrogen starvation. Cells were collected at the indicated time points after induction of nitrogen starvation and were used for immunoblotting.

Figure 2 with 2 supplements
Atg43 is a transmembrane protein localized to the MOM.

(A, B) Wild-type cells were grown in EMM and shifted to EMM–N for nitrogen starvation. Cells were collected at the indicated time points after shifting to nitrogen starvation medium and were then subjected to immunoblotting. (C) The indicated strains were grown in EMM at 26°C. The temperature was then shifted to 37°C to inactivate TORC1. Cells were collected at the indicated time points after the temperature shift and were used for immunoblotting. (D, E) The indicated strains were collected at the indicated time points after shifting to nitrogen starvation medium and were used for immunoblotting. (F) Cells expressing GFP-Atg43 and Tuf1-mRFP were grown in EMM (+N) or cultured in EMM–N for 12 hr (–N) for microscopy. A magnified view of the indicated area is shown in the right panel. Scale bars represent 5 μm. BF, bright-field image. (G) Fractionation was conducted using cells expressing Tuf1-mRFP. The total cell homogenate (total) was fractionated by centrifugation to obtain a mitochondria-enriched pellet (mit) and supernatant (sup). Tuf1-mRFP and actin were detected as markers of the mitochondria and cytosol, respectively. (H) A mitochondrial fraction was prepared from cells expressing Tom70-GFP, Mic60-FLAG, and Tuf1-mRFP, treated with (+) or without (–) proteinase K, under different conditions. Hypo-osmotic swelling resulted in rupture of the MOM and the detergent Triton X-100 lysed mitochondria. Tom70-GFP, Mic60-FLAG, and Tuf1-mRFP were detected as markers of the mitochondrial outer membrane, inner membrane, and matrix, respectively. (I) The mitochondrial fraction (mit) was prepared from cells expressing Tom70-GFP, treated with sodium carbonate, and separated into the soluble supernatant (sup) and membrane pellet (pellet) by centrifugation. Tom70-GFP and Atp2 were detected as markers for integral and peripheral membrane proteins, respectively. (J) The mitochondrial fraction was prepared from cells expressing N-terminal or C-terminal FLAG-tagged Atg43 (FLAG-Atg43 or Atg43-FLAG, respectively), followed by treatment with (+) or without (–) proteinase K. The Triton X-100 detergent lysed mitochondria. The proteinase-resistant band is indicated by the arrowhead. Histone H3 was used as a loading control (LC) for immunoblotting.

Figure 2—figure supplement 1
The C-terminal region of Atg43 is conserved.

(A) A multiple sequence alignment of Atg43 proteins in the Schizosaccharomyces species. The alignment was produced using ClustalW and modified manually. The N-terminal AIM is indicated. (B) Alignment of fungal sequences similar to the C-terminal region of Atg43. The alignment was produced using ClustalW and modified manually. The predicted C-terminal transmembrane domain is indicated.

Figure 2—figure supplement 2
Atg43 is a transmembrane protein that is expressed during nitrogen starvation.

(A) The indicated strains were grown in EMM and the temperature was shifted to 37°C to inactivate TORC1. Cells were collected at the indicated time points after the temperature shift and were used for immunoblotting. (B) Wild-type cells and cells expressing Atg43 under the thiamine-repressible nmt1 promoter were grown in EMM containing thiamine for moderate expression. Cells were collected at the indicated time points after shifting to nitrogen starvation medium and were used for immunoblotting. Histone H3 was used as a loading control (LC). (C) Schematic representation of the structure of Atg43. The region conserved among fungi is highlighted in blue. A C-terminal predicted transmembrane domain (TM) is indicated. (D) Cells expressing the C-terminal 80 aa of Atg43 (C80) tagged with FLAG at the N-terminus (FLAG-C80) or C-terminus (C80-FLAG) were grown in YES. The mitochondrial fraction was prepared and treated with (+) or without (–) proteinase K. The detergent Triton X-100 lysed mitochondria. The proteinase-resistant band is indicated by an arrowhead.

Figure 3 with 1 supplement
Atg43 contains an AIM that is essential for mitophagy.

(A) Schematic representation of N-terminal deleted forms of Atg43. The region conserved among fungi is highlighted in blue. The N-terminal AIM and C-terminal predicted transmembrane domain (TM) are indicated. (B) Wild-type cells and strains expressing FLAG-tagged Atg43, with or without N-terminal truncation, under the thiamine-repressible nmt1 promoter, were grown in EMM containing thiamine for moderate expression. Cells were collected at the indicated time points after shifting to nitrogen starvation medium and were subsequently used for immunoblotting. (C) Cells expressing wild-type and AIM-mutated (ΔAIM) Atg43 from the native gene locus were collected at the indicated time points after shifting to nitrogen starvation medium and subjected to immunoblotting. (D, E) A yeast two-hybrid assay between activation domain (AD)-fused Atg8 and DNA-binding domain (BD)-fused Atg43 fragments, without or with mutations in the N-terminal AIM (ΔAIM). (F, G) Crude cell lysates (input) were prepared from cells with (+) or without (–) expression of GFP-fused Atg8 and FLAG-tagged Atg43 or AIM-mutated Atg43 (ΔAIM). Anti-GFP immunoprecipitants (IP: GFP) were analyzed by immunoblotting. Cells were collected 12 hr after shifting to nitrogen starvation medium and were subjected to immunoprecipitation. (H) Cells expressing wild-type and deleted forms of Atg43 from the native gene locus were collected at the indicated time points after shifting to nitrogen starvation medium and were subsequently used for immunoblotting. (I) The indicated strains were grown in EMM, and their serial dilutions were spotted onto solid YES medium for the growth assay. (J, K) Cells expressing N-terminal truncated forms of Atg43 tagged with GFP were grown in EMM for microscopy. Tuf1-mRFP was detected as a mitochondrial marker. Scale bars represent 5 μm. BF, bright-field image. Histone H3 was used as a loading control (LC) for immunoblotting.

Figure 3—figure supplement 1
The N-terminal region of Atg43 interacts with Atg8 and is dispensable for the mitochondrial localization.

(A) Immunoprecipitation experiments were conducted using crude cell lysates (input) prepared from FLAG-Atg43-expressing cells with co-expression of the indicated proteins. Cells were collected 12 hr after induction of nitrogen starvation and subjected to immunoprecipitation. (B) The atg43-1 allele was chromosomally tagged with FLAG (atg43-1:FLAG) and the expressed protein was detected by immunoblotting in parallel with the untagged atg43-1 (atg43-1) and tagged wild-type (atg43:FLAG) strains. Histone H3 was used as a loading control (LC). (C) Fractionation was conducted using atg43-1:FLAG cells. The cell homogenate was fractionated by centrifugation to obtain a mitochondria-enriched pellet (mit) and supernatant (sup). Tuf1-mRFP and actin were detected as markers of the mitochondria and cytosol, respectively.

The C-terminal region of Atg43 that contains a predicted transmembrane domain is required for mitochondrial localization.

(A) Schematic representation of C-terminal deleted forms of Atg43. The region conserved among fungi is highlighted in blue. The N-terminal AIM and predicted C-terminal transmembrane domain (TM) are indicated. (B) Cells expressing wild-type and C-terminal deleted forms of Atg43 from the native gene locus were collected at the indicated time points after shifting to nitrogen starvation medium and were subjected to immunoblotting. Each strain contained an integration vector expressing the mitophagy-defective form of Atg43, that lacks the 164 N-terminal aa, to maintain a normal growth rate. (C) The indicated strains were grown in EMM, and their serial dilutions were spotted onto solid YES medium for a growth assay. (D) Cells expressing wild-type and C-terminal truncated forms of GFP-tagged Atg43 were grown in EMM for microscopy. Tuf1-mRFP was detected as a mitochondrial marker. (E) Wild-type (fis1+) and GBP-fis1 cells co-expressing GFP-tagged Atg43 with a 60 aa deletion in the C-terminus and Tuf1-mRFP were grown in EMM for microscopy. (F) Wild-type (fis1+) and GBP-fis1 cells expressing full-length (wild-type) or C-terminal truncated Atg43 (Atg40ΔC60) with or without GFP fusion were collected at the indicated time points after shifting to nitrogen starvation medium. Cells were then used for immunoblotting. (G) Cells expressing the GFP-fused aa 185–224 of Atg43 and Tuf1-mRFP were grown in EMM for microscopy. A magnified view of the indicated area is shown in the right panel. (H) Mitophagy-defective atg43-1 cells with GBP-fused Fis1 were transformed to express GFP, GFP-fused aa 21–40 of Atg43, or GFP-fused Atg8. The indicated strains were collected at the indicated time points after shifting to nitrogen starvation medium and used for immunoblotting. Histone H3 was used as a loading control (LC) for immunoblotting. Scale bars represent 5 μm. BF, bright-field image.

Figure 5 with 1 supplement
The MIM complex facilitates mitophagy through the loading of Atg43 to the MOM.

(A, B) Crude cell lysates (input) were prepared from cells co-expressing GFP-fused Mim2 or Mim1 with (+) or without (–) FLAG-tagged Atg43. Anti-FLAG immunoprecipitants (IP: FLAG) were analyzed by immunoblotting. (C, E) The indicated strains co-expressing GFP-Atg43 and Tuf1-mRFP were grown in EMM for microscopy. (D) The indicated strains expressing Tuf1-mRFP were collected at the indicated time points after shifting to nitrogen starvation medium and were used for immunoblotting. (F) The indicated strains co-expressing a GFP-fused C-terminal truncated form of Atg43 (Atg43ΔC60-GFP) and Tuf1-mRFP were grown in EMM for microscopy. (G) Wild-type, mim1Δ, mim2Δ, and tom70Δ cells co-expressing GBP-fused Fis1 and GFP-fused Atg43 lacking the 60 C-terminal aa (Atg43ΔC60-GFP) were collected at the indicated time points after shifting to nitrogen starvation medium. Cells were then used for immunoblotting. Histone H3 was used as a loading control (LC) for immunoblotting. Scale bars represent 5 μm. BF, bright-field image.

Figure 5—figure supplement 1
The MIM complex facilitates the mitochondrial localization of Atg43 and Tom70.

(A) Identification of Mim2 as an Atg43-binding protein. Mass spectrometric analysis of proteins co-purified with the 80 C-terminal aa of Atg43 with a FLAG tag was conducted. Wild-type cells expressing untagged Atg43 were used as a negative control. The value (score) represents the relative protein abundance as expressed by spectral abundance factor (SAF). Mim2 was identified along with 37 peptide-spectrum matches, at a targeted false discovery rate (FDR) of 1.0% at the peptide level. (B–E) Atg43 interacts with the MIM complex. Immunoprecipitation experiments were conducted using crude cell lysates (input) prepared from cells with (+) or without (–) expression of the indicated proteins. Anti-FLAG (IP: FLAG) and anti-GFP (IP: GFP) immunoprecipitants were analyzed by immunoblotting. (F) The indicated strains were grown in EMM, and their serial dilutions were spotted onto solid YES medium for the growth assay. (G) Atg43 is unstable in the absence of the MIM complex. The indicated strains co-expressing Tom70-GFP and Tuf1-mRFP were grown in EMM for immunoblotting. Histone H3 was used as a loading control (LC). (H, I) Mitochondrial localization of Tom70 requires the MIM complex but does not require Atg43. The indicated strains co-expressing GFP-tagged Tom70 with Tuf1-mRFP were grown in EMM for microscopy. (J, K) Mitochondrial localization of the MIM complex is independent of Atg43 and Tom70. The indicated strains co-expressing Tuf1-mRFP with GFP-tagged Mim1 (J) or Mim2 (K) were grown in EMM for microscopy. Scale bars denote 5 μm. BF, bright-field image.

Figure 6 with 1 supplement
C-terminal region of Atg43 is required for its interaction with the MIM complex.

(A, C) Schematic representation of deleted forms of Atg43. The region conserved among fungi is highlighted in blue. The C-terminal predicted transmembrane domain (TM) is also indicated. (B, D) Crude cell lysates (input) were prepared from cells co-expressing GFP-fused Mim2 and FLAG-tagged Atg43 with or without deletion. Anti-FLAG immunoprecipitants (IP: FLAG) were analyzed by immunoblotting. (E) Schematic representation of the C-terminal region of Atg43. (F) Schematic model describing the loading and function of Atg43 on the MOM.

Figure 6—figure supplement 1
Atg43 interacts with the MIM complex.

(A, B) Immunoprecipitation experiments were conducted using crude cell lysates (input) prepared from cells with (+) or without (–) expression of the indicated proteins. Anti-FLAG (IP: FLAG) and anti-GFP (IP: GFP) immunoprecipitants were analyzed by immunoblotting.

Mitophagy maintains growth ability during nitrogen starvation.

(A–C) The indicated strains were grown in EMM and shifted to EMM–N. Cells were collected at the indicated time points after induction of nitrogen starvation and spotted onto solid YES medium for the growth assay. Cells auxotrophic for leucine, uracil, histidine, and lysine (leu1-32, ura4, his7, lys1) were used in (B). (D, E) Cells were collected at the indicated time points after induction of nitrogen starvation and stained with MitoSOX (D) or Mito Tracker (E). Sdh2-GFP was detected as a mitochondrial marker. Scale bars represent 5 μm. BF, bright-field image. (F) Serial dilutions of the indicated strains collected immediately before or 5 days after induction of nitrogen starvation were spotted onto glycerol plates. (G) Schematic model for mitophagy mediated by Atg43 on the MOM.

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (S. pombe)972Laboratory stockATCC24843Stocked in T. Kanki lab.
Strain, strain background (S. pombe)CA101Laboratory stockh- leu1-32
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)CA103Laboratory stockh- ura4-D18
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)MM72-1DNBPR (https://yeast.nig.ac.jp/yeast/)FY7652h- leu1-32 ura4-D18
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP407This studyh- ura4-D18 tuf1-mRFP::ura4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1079This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1669This studyh- ura4-D18 tuf1-mRFP::ura4 tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP669This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3834This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::nat atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1013This studyh- atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1057This studyh- ura4-D18 tuf1-mRFP::ura4 atg43Δ::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3353This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4 pNATZA31
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1941This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4 pNATZA31-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3473This studyh- atg43Δ::kan pNATZA31
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP2705This studyh- atg43Δ::kan pNATZA31-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP183This studyh- ura4-D18 GFP-atg8::ura4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP991This studyh- ura4-D18 GFP-atg8::ura4 atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP743This studyh- pgk1-mEGFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP989This studyh- pgk1-mEGFP::hph atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP671This studyh- ura4-D18 tuf1-mRFP::ura4 yop1-mEGFP::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP987This studyh- ura4-D18 tuf1-mRFP::ura4 yop1-mEGFP::nat atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP25This studyh- sdh2-mEFGP::kan tuf1-mRFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP137This studyh- sdh2-mEFGP::kan tuf1-mRFP::hph atg1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP145This studyh- sdh2-mEFGP::kan tuf1-mRFP::hph isp6Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1273This studyh- tom70-mEGFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)CA1258This studyh- tuf1-mRFP::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP78This studyh- tuf1-mRFP::kan fun14Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1321This studyh- tom70-mEGFP::hph atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3475This studyh- mic60-mEGFP::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3477This studyh- mic60-mEGFP::nat atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP209This studyh- atg1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP271This studyh- isp6Δ::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP667This studyh- ura4-D18 tdh1-mRFP::ura4 sdh2-mEGFP::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP985This studyh- ura4-D18 tdh1-mRFP::ura4 sdh2-mEGFP::nat atg43-1::kanMX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1677This studyh- ura4-D18 GFP-atg8::ura4 tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1675This studyh- pgk1-mEGFP::kan tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1679This studyh- ura4-D18 tuf1-mRFP::ura4 yop1-mEGFP::nat tom70Δ::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)CA9659DOI:10.1111/j.1365–2443.2006.01025.xh+ ura4-D18 tor2-13::ura4
Strain, strain background (S. pombe)TFSP1539This studyh- atg7Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1071This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3237This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43 atg7Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3177This studyh- tom70-mEGFP::hph mic60-FLAG::kan tuf1-mRFP::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1097This studyh- hph::P3nmt1:FLAG-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1717This studyh- kan::P3nmt1:atg43-FLAG::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP2633This studyh- hph::P3nmt1:FLAG-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3593This studyh- kan::P3nmt1:atg43(C80)-FLAG::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)CA12620This studyh+ ura4-D18 tor2-13::ura4 tuf1-mRFP::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1399This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P3nmt1:atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1101This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P3nmt1:FLAG-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3205This studyh- ura4-D18 tuf1-mRFP::ura4?hph::P3nmt1:FLAG-atg43ΔN20
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3317This studyh- ura4-D18 tuf1-mRFP::ura4?hph::P3nmt1:FLAG-atg43ΔN40
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3651This studyh- ura4-D18 tuf1-mRFP::ura4 atg43ΔAIM::nat
Stocked in T. Kanki lab.
Strain, strain background
(S. pombe)
TFSP3979This studyh- hph::P3nmt1:FLAG-atg43 kan::P3nmt1:GFP-atg8
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3981This studyh- hph::P3nmt1:FLAG-atg43ΔAIM kan::P3nmt1:GFP-atg8
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP2721This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3291This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph atg43ΔN41-80::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3293This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph atg43ΔN81-120::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3307This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph atg43ΔN121-164::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3289This studyh- atg43ΔN164::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3315This studyh- atg43ΔN184::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1107This studyh- atg43Δ::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3261This studyh- ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80) hph::P81nmt1:GFP-atg43ΔN164
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4041This studyh- hph::P3nmt1:FLAG-atg43 pNATZA1-GFP
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3983This studyh- hph::P3nmt1:FLAG-atg43 pNATZA1-GFP-atg8
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4263This studyh- hph::P3nmt1:FLAG-atg43 pNATZA1-GFP-atg11
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4265This studyh- hph::P3nmt1:FLAG-atg43 pNATZA1-atg11-GFP
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1461This studyh- atg43-1::kanMX4-FLAG::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1021This studyh- atg43-FLAG::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4289This studyh- tuf1-mRFP::nat atg43-1::kanMX4-FLAG::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3263This studyh- ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80) hph::P81nmt1:GFP-atg43ΔN184
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3223This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph pNATZA31-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3241This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph pNATZA31-atg43(C80) atg43ΔC20::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3253This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph pNATZA31-atg43(C80) atg43ΔC60::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3305This studyh- ura4-D18 tuf1-mRFP::ura4 sdh2-mEGFP::hph pNATZA31-atg43(C80) atg43ΔC61-80::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP2419This studyh- atg43ΔC20::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3287This studyh- atg43ΔC60::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3363This studyh- atg43ΔC61-80::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3225This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43 pNATZA31-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3255This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43ΔC20::kan pNATZA31-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3259This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43ΔC60::kan pNATZA31-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3343This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43ΔC61-80::kan pNATZA31-atg43(C80)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3559This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 atg43ΔC60-mEGFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3529This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 kan::P3nmt1:GBP-fis1 atg43ΔC60-mEGFP::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3453This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3557This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 kan::P3nmt1:GBP-fis1 atg43ΔC60::hph
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3803This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4 hph::P3nmt1:GBP-fis1 pNATZA1-GFP
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3793This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4 hph::P3nmt1:GBP-fis1pNATZA1-GFP-atg43(N21-40)
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3785This studyh- ura4-D18 tuf1-mRFP::ura4 atg43-1::kanMX4 hph::P3nmt1:GBP-fis1 pNATZA1-GFP-atg8
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3165This studyh- kan::P3nmt1:GFP-mim2
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3163This studyh- kan::P3nmt1:GFP-mim2 hph::P3nmt1:FLAG-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3215This studyh- kan::P3nmt1:GFP-mim1
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3213This studyh- kan::P3nmt1:GFP-mim1 hph::P3nmt1:FLAG-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3247This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43 mim1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3173This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43 mim2Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3283This studyh- ura4-D18 tuf1-mRFP::ura4 mim1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3271This studyh- ura4-D18 tuf1-mRFP::ura4 mim2Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1779This studyh- ura4-D18 tuf1-mRFP::ura4 hph::P81nmt:GFP-atg43 tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3565This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 kan::P3nmt1:GBP-fis1 atg43ΔC60-mEGFP::hph mim1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3567This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 kan::P3nmt1:GBP-fis1 atg43ΔC60-mEGFP::hph mim2Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3569This studyh- leu1-32 ura4-D18 tuf1-mRFP::ura4 pNATZA31-atg43(C80)::leu1MX4 kan::P3nmt1:GBP-fis1 atg43ΔC60-mEGFP::hph tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3217This studyh- kan::P3nmt1:GFP-mim2hph::P3nmt1:FLAG-mim1
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3761This studyh- mim1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3763This studyh- mim2Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3161This studyh- ura4-D18 tuf1-mRFP::ura4 tom70-mEGFP::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3459This studyh- ura4-D18 tuf1-mRFP::ura4 tom70-mEGFP::kan mim1Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3455This studyh- ura4-D18 tuf1-mRFP::ura4 tom70-mEGFP::kan mim2Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3479This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim1
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3523This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim1 atg43Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3563This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim1tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3175This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim2
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3525This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim2 atg43Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3561This studyh- ura4-D18 tuf1-mRFP::ura4 kan::P3nmt1:GFP-mim2 tom70Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3485This studyh- kan::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) hph::P3nmt1:FLAG-atg43
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3487This studyh- kan::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) hph::P3nmt1:FLAG-atg43ΔN164
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3489This studyh- kan::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) hph::P3nmt1:FLAG-atg43ΔN184
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3601This studyh- leu1-32 hph::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) leu1MX4::P3nmt1:FLAG-atg43ΔC61-80::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3617This studyh- leu1-32 hph::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) leu1MX4::P3nmt1:FLAG-atg43ΔC20::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3603This studyh- leu1-32 hph::P3nmt1:GFP-mim2 pNATZA31-atg43(C80) leu1MX4::P3nmt1:FLAG-atg43ΔC60::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3201This studyh- tom70-FLAG::hph kan::P3nmt1:GFP-mim2
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3233This studyh- hph::P3nmt1:FLAG-atg43 tom70-mEGFP::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP3649This studyh- atg43ΔAIM::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)MKY1NBPR (https://yeast.nig.ac.jp/yeast/)FY7455h+ leu1-32 ura4 his7 lys1
Strain, strain background (S. pombe)TFSP3481This studyh+ leu1-32 ura4 his7 lys1 atg7Δ::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4259This studyh+ leu1-32 ura4 his7 lys1 atg43ΔAIM::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP1559This studyh- isp6Δ::hph atg7Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4255This studyh- isp6Δ::hph atg43ΔAIM::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP10This studyh- sdh2-mEFGP::kan
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4271This studyh- sdh2-mEFGP::kan atg7Δ::nat
Stocked in T. Kanki lab.
Strain, strain background (S. pombe)TFSP4273This studyh- sdh2-mEFGP::kan atg43ΔAIM::nat
Stocked in T. Kanki lab.
Antibodyanti-RFP (Rabbit polyclonal)MBLCat# PM005, RRID:AB_591279WB (1:3000)
Antibodyanti-GFP (Mouse monoclonal)Takara BioCat# 632380, RRID:AB_10013427WB (1:10000)
Antibodyanti-histone H3 (Rabbit polyclonal)AbcamCat# ab1791, RRID:AB_302613WB (1:10000)
Antibodyanti-FLAG (Mouse monoclonal)SigmaCat# F1804, RRID:AB_262044WB (1:4000)
Antibodyanti-DDDDK (Rabbit polyclonal)MBLCat# PM020, RRID:AB_591224WB (1:4000)
Antibodyanti-Atp2 (Rabbit polyclonal)AbcamCat# ab128743, RRID:AB_2810299WB (1:2000)
Antibodyanti-Actin (Mouse monoclonal)AbcamCat# ab8224, RRID:AB_449644WB (1:4000)
Antibodyanti-mouse IgG (Goat polyclonal, Peroxidase conjugated)Merck MilliporeCat# AP124P, RRID:AB_90456WB (1:10000)
Antibodyanti-rabbit IgG (Goat polyclonal, Peroxidase conjugated)Jackson ImmunoResearchCat# 111-035-003, RRID:AB_2313567WB (1:10000)
Antibodyanti-guineapig IgG (Goat polyclonal, Peroxidase conjugated)Jackson ImmunoResearchCat# 106-035-003, RRID:AB_2337402WB (1:10000)
Antibodyanti-FLAG beadsSigmaCat# M8823, RRID:AB_2637089
Antibodyanti-GFP beadsChromotekCat# gtma, RRID:AB_2631358
Antibodyanti-Atg43 (Guineapig polyclonal)This studyWB (1:4000)
Stocked in T. Kanki lab.
Recombinant DNA reagentpFA6a-leu1MX4 (plasmid)NBPRFYP2892
Recombinant DNA reagentpJQW#600 (plasmid)NBPRFYP3955
Recombinant DNA reagentpNATZA1 (plasmid)NBPRFYP2874
Recombinant DNA reagentpNATZA31 (plasmid)NBPRFYP2879
Recombinant DNA reagentpNATZA31-atg43 (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA31-atg43(C80) (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA1-GFP (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA1-GFP-atg8 (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA1-GFP-atg11 (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA1-atg11-GFP (plasmid)This studyStocked in T. Kanki lab.
Recombinant DNA reagentpNATZA1-GFP-atg43(N21-40) (plasmid)This studyStocked in T. Kanki lab.
Commercial assay or kitEzWestLumi OneAttoWSE-7110
Commercial assay or kitEzWestLumi plusAttoWSE-7120
Commercial assay or kitClarity Max Western ECL SubstrateBio-Rad1705062
Chemical compound, drugMitoTracker Red CMXRosThermo Fisher ScientificM7512(250 nM)
Chemical compound, drugMito SOX RedThermo Fisher ScientificM36008(5 μM)
Software, algorithmImage LabBio-Rad
Software, algorithmMetaMorph 7Molecular Devices

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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