Metabolic stresses that stimulate acute mitochondrial biogenesis induce MDC formation.

A. Time-course immunoblot of mitochondrial protein levels in wild-type yeast during glucose restriction (0-6 h). Por1, Om45, Aco1, and Cit1 were detected with the indicated antibodies; Vph1 and Ponceau staining serve as loading controls. B. Super-resolution images of wild-type cells grown in glucose-replete (YPAD) or glucose-restricted (YPA) media for 3 h. Cells express Tom70-GFP and Tim50-mCherry. Line-scan intensity profiles are shown below and correspond to the yellow dashed lines. The white arrow indicates an MDC. Scale bar, 2 μm. C. Quantification of the percentage of cells containing MDCs following 3 h glucose restriction. D. Immunoblot analysis of GFP processing after 6 h of the indicated treatments in wild-type, pep4Δ, dnm1Δ, and atg5Δ strains expressing Tom70-GFP, or wild-type cells expressing Tim50-GFP. Free GFP indicates proteolytic cleavage. Ponceau staining serves as a loading control. E. Quantification of MDC formation following a 3 h carbon-source shift. Cells were grown overnight in glucose (YPAD) and shifted to media containing the indicated carbon sources: glucose (Glc), no added carbon source (-), glycerol (Glyc), galactose (Gal), sucrose (Suc), raffinose (Raf), or fructose (Fru). F. Immunoblot analysis of mitochondrial protein levels following glucose restriction, carbon-source switching, glycolytic inhibition, or salt stress (0.8 M NaCl or 1 M KCl). Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. G. Quantification of MDC formation after 3 h of salt stress (0.8 M NaCl or 1 M KCl). Quantification and statistics. For all MDC measurements, n = 3 independent experiments with 100 cells scored per replicate; bars show mean ± SEM.

MDC formation during glucose restriction requires trace glucose but is independent of amino-acid availability.

A. Quantification of the percentage of cells containing MDCs following glucose withdrawal in different media conditions. Cells were grown overnight in YPAD or synthetic (SD) media and shifted to the indicated media with or without glucose for 2 h. B. Super-resolution images of cells following glucose starvation (0% glucose, S) or glucose restriction (0.02% glucose, S + 0.02% glucose) for 2 h in synthetic media. Cells express Tom70-GFP and Tim50-mCherry. White arrows indicate MDCs. Scale bar, 5 μm. C. Quantification of MDC formation following 2 h add-back treatments. Cells were grown overnight in YPAD and shifted to the indicated conditions: YPAD (control), YPA (glucose restriction), S (no glucose), S + casamino acids (CasAA), S + peptone, S + yeast extract, or S + 0.02% glucose. D. Quantification of MDC formation across a glucose gradient. Cells were grown overnight in YPAD and shifted for 2 h to synthetic media containing the indicated glucose concentrations; YPAD and YPA serve as controls. E. Correlation analysis of metabolite log₂-fold changes following 2 h treatments. Left: glucose restriction (YPAD→YPA) versus glucose starvation (YPAD→S). Right: glucose restriction (YPAD→YPA) versus glycerol shift (YPAD→YPAG). Red points represent TCA cycle metabolites; blue, amino acid–related metabolites; dark blue, branched-chain amino acid–related metabolites; and black, other metabolites. Correlation coefficients (R2) are indicated. F. Analysis of central carbon metabolism metabolites following 2 h treatments (YPAD, YPA, YPAG, S). Values are shown as linear fold change relative to YPAD. n = 4; error bars represent mean ± SEM. G. Analysis of methionine and polyamine pathway metabolites under the same conditions as in (F). n = 4; error bars represent mean ± SEM. Quantification and statistics. Microscopy-based assays were performed with n = 3 independent experiments and 100 cells scored per replicate; error bars represent mean ± SEM. Statistical analysis for (F) and (G) was performed using two-way ANOVA with Holm–Šídák multiple-comparisons testing. False discovery rate (FDR) correction (q = 0.05) was applied.

Snf1 is required for glucose restriction– and salt stress-induced MDCs.

A. Widefield images of wild-type and snf1Δ cells following 2 h glucose restriction. Cells express Tom70-GFP and Tim50-mCherry. White arrows indicate MDCs. Scale bar, 5 μm. B. Quantification of MDC formation in wild-type and snf1Δ cells following 3 h carbon-source shifts. Cells were grown overnight in glucose (YPAD) and shifted to media containing glucose (Glc), no added carbon source (-), galactose (Gal), or glycerol (Glyc). C. Schematic of glucose sensing and repression through the Snf1 pathway. D. Quantification of MDC formation in wild-type and snf1Δ cells following 3 h salt stress (0.8 M NaCl or 1 M KCl). Wild-type and snf1Δ cells were analyzed in parallel. Wild-type data are reproduced from Figure 1G to facilitate direct comparison with the mutant. E. Immunoblot analysis of Snf1-AID-FLAG degradation following auxin treatment. Control (Snf1-AID-FLAG) and test (Snf1-AID-FLAG + His3-OsTIR1) strains were treated with DMSO or auxin for the indicated times. FLAG was detected by immunoblot; Ponceau staining serves as a loading control. F. Quantification of MDC formation following auxin-mediated Snf1 depletion. Cells were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to glucose restriction for 2 h. G. Quantification of MDC formation in Snf1-AID strains following auxin-mediated Snf1 depletion. Cells were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to salt stress for 3 h. H. Quantification of MDC formation in wild-type and snf1Δ cells following 2 h rapamycin treatment. I. Quantification of MDC formation in Snf1-AID strains following auxin-mediated Snf1 depletion. Cells were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to rapamycin treatment for 2 h. Quantification and statistics. All MDC measurements (B, D, F-I) were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

Mig1 derepression is required downstream of Snf1 for MDC induction.

A. Schematic of the glucose repression pathway in yeast. B. Widefield images of wild-type, snf1Δ, mig1Δ, and snf1Δ mig1Δ cells following 2 h glucose restriction. Cells express Tom70-GFP and Tim50-mCherry. White arrows indicate MDCs. Scale bar, 5 μm. C. Quantification of MDC formation in wild-type, snf1Δ, and snf1Δ mig1Δ cells following 2 h glucose restriction. D. Quantification of MDC formation in wild-type, snf1Δ, and snf1Δ mig1Δ cells following 3 h salt stress (0.8 M NaCl or 1 M KCl). Wild-type and snf1Δ cells were analyzed in parallel with snf1Δ mig1Δ cells. The wild-type and snf1Δ data are reproduced from Figures 1G and 3D, respectively, to facilitate direct comparison. E. Quantification of MDC formation in wild-type cells following glucose restriction or NaCl stress in the presence or absence of thiolutin. Cells were treated for 2 h under glucose restriction conditions or for 3 h under NaCl stress. F. Quantification of MDC formation in snf1Δ MIG1-AID-FLAG cells with or without integrated HIS3::GPD-OsTIR1. Cells were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to glucose restriction for 2 h. G. Quantification of MDC formation in mig1Δ SNF1-AID-FLAG cells with or without integrated HIS3::GPD-OsTIR1. Cells were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to glucose restriction for 2 h. Quantification and statistics. All MDC measurements were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

Transient induction of mitochondrial-targeted protein expression is a common theme among MDC inducers and sufficient to stimulate MDC formation.

A. Immunoblot analysis of mitochondrial protein levels in wild-type, snf1Δ, mig1Δ, and snf1Δ mig1Δ cells following glucose restriction (3 h) or salt stress (0.8 M NaCl or 1 M KCl, 4 h). Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. B. Mitochondrial proteomics analysis of Hap4 overexpression (OE) versus empty vector (EV). Top: volcano plot showing differentially regulated proteins using cutoffs of ≥1.5-fold change and FDR-adjusted p ≤ 0.01. Bottom: Gene Ontology (GO) enrichment analysis of upregulated proteins. C. Schematic of the β-estradiol–inducible Hap4 expression system. D. Immunoblot analysis of Hap4 induction following â-estradiol treatment. Strains expressing GAL1-HAP4-FLAG or GAL1-HAP4-FLAG + GAL4-ERT2 were treated with DMSO or â-estradiol as indicated. FLAG, Cit1, Om45, Por1, and Aco1 were detected; Pgk1, Vph1, and Ponceau staining serve as loading controls. The asterisk indicates a nonspecific band. E. Quantification of MDC formation in Hap4 expression strains following 3.5 h β-estradiol treatment at the indicated concentrations. Cells were maintained in SD-URA supplemented with casamino acids. F. Quantification of MDC formation in Hap4 expression strains under glucose-replete and glucose-restricted conditions with β-estradiol treatment. Cells were grown overnight in SD-URA and shifted to SD-URA, S-URA + 0.02% glucose, YPAD, or YPA as indicated for 3.5 h. Quantification and statistics. All MDC measurements were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

MDC formation requires Tom70/Tom71-dependent mitochondrial protein targeting.

A. Quantification of MDC formation in wild-type, tom70Δ, and tom70Δ tom71Δ cells following 2 h glucose restriction. Cells express Tcd2-GFP and Tim50-mCherry. B. Quantification of MDC formation in wild-type and tom70Δ tom71Δ cells following 2 h glucose restriction or rapamycin treatment. Cells express Cox7-GFP and Tim50-mCherry. C. Quantification of the percentage of cells exhibiting ER-localized Oac1-GFP, as shown in (D), determined by colocalization with Sec61-mCherry. Cells were treated for 3 h in YPAD (control) or YPA (glucose restriction). D. Widefield images of Oac1-GFP localization in wild-type and tom70Δ tom71Δ cells following 3 h glucose restriction. Cells express Oac1-GFP and Sec61-mCherry. Line-scan intensity profiles corresponding to the yellow dashed lines are shown. Scale bar, 5 μm. E. Quantification of the percentage of cells exhibiting cytosolic Om45–GFP, as shown in (F), defined by diffuse cytosolic GFP signal. Cells were treated for 3 h in YPAD (control) or YPA (glucose restriction). F. Widefield images of Om45-GFP localization in wild-type and tom70Δ tom71Δ cells following 3 h glucose restriction. Cells express Om45–GFP with Tom70-mCherry or Tim50-mCherry. Line-scan intensity profiles corresponding to the yellow dashed lines are shown. Scale bar, 5 μm. G. Immunoblot analysis of mitochondrial protein levels in wild-type and tom70Δ tom71Δ cells following 3 h growth in YPAD or YPA. Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. H. Proposed unified model for MDC formation. Snf1-dependent metabolic remodeling stresses, Snf1-independent nutrient/vacuolar stresses, and direct protein-load inputs converge on increased hydrophobic membrane-protein load at the OMM during acute mitochondrial remodeling. Tom70/Tom71-dependent targeting promotes engagement of these cargos with mitochondria, triggering MDC formation and cargo sequestration when protein load exceeds the adaptive capacity of the organelle. MDC cargos are subsequently delivered to the vacuole for degradation. Quantification and statistics. All microscopy-based assays were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

Characterization of MDCs induced by mitochondrial biogenesis–activating conditions (related to Figure 1).

A. Immunoblot time course of mitochondrial protein levels in wild-type cells grown in glucose-replete conditions (YPAD) over 0-6 h. Por1, Om45, Aco1, and Cit1 are shown; Vph1 and Ponceau staining serve as loading controls. B. Quantification of MDC diameter under different induction conditions. Wild-type diploid cells were treated with glucose restriction (YPA), rapamycin (RAP), or concanamycin A (ConcA). C. Widefield images of MDC cargo colocalization following 2 h glucose restriction. Cells express Tom70–mCherry to label MDCs and the indicated GFP-tagged proteins (Tim50, Tcd2, Mcp1, Cox7, Oac1, or Dic1). White arrows indicate MDCs. Scale bar, 5 μm. D. Quantification of MDC cargo colocalization, as shown in (C). Colocalization was scored when MDCs overlapped with the indicated GFP signal. E. Immunoblot analysis of GFP processing for MDC cargo proteins following glucose restriction for the indicated times. Strains expressing Mir1-GFP, Tcd2-GFP, or Dic1-GFP were analyzed. Free GFP indicates proteolytic cleavage; GFP was detected by immunoblot, and Vph1 and Ponceau staining serve as loading controls. F. Quantification of MDC formation following 2 h treatment with 2-deoxy-D-glucose (2-DG) at the indicated concentrations. Quantification and statistics. For MDC frequency measurements, n = 3 independent experiments with 100 cells scored per replicate; for diameter measurements (B), n = 100 MDCs per condition. Error bars represent mean ± SEM.

Further characterization of the trace-glucose requirement and amino acid independence of MDC formation (related to Figure 2).

A. Quantification of MDC formation following 3 h carbon-source shifts in synthetic media. Cells were grown overnight in synthetic media with 2% glucose and shifted to media containing glucose (Glc), no added carbon source (-), glycerol (Glyc), galactose (Gal), sucrose (Suc), raffinose (Raf), or fructose (Fru). B. Quantification of the percentage of cells exhibiting fragmented mitochondria (see 2B and 2D). C. Quantification of MDC formation following 2 h cycloheximide (CHX) treatment under varying glucose and amino acid conditions. Cells were grown overnight in synthetic media and shifted to the indicated conditions. D. Quantification of mitochondrial fragmentation in wild-type and dnm1Δ cells under different media conditions. Cells were grown overnight in YPAD and shifted for 2 h to YPAD, synthetic media with 0.02% glucose (S + 0.02% glucose), synthetic media without carbon (S), or synthetic media with glycerol (S + glycerol). E. Quantification of MDC formation in wild-type and dnm1Δ cells under the conditions described in (D). F. Analysis of whole-cell amino acid metabolites following 2 h treatments (YPAD, YPA, YPAG, S), shown as linear fold change relative to YPAD. G. Quantification of MDC formation under trace-glucose conditions with or without amino acid supplementation. Prototrophic cells were grown overnight in YPAD, synthetic (SD), or minimal media and shifted for 2 h to the indicated conditions, including glucose restriction (0.02% glucose) and casamino acid supplementation. H. Quantification of MDC formation under trace-glucose conditions in minimal or nitrogen-starvation media. Cells were grown overnight in minimal media with 2% glucose and shifted for 2 h to the indicated conditions. Quantification and statistics. Microscopy-based assays (A–E, G–H) were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical analysis for (F) was performed using two-way ANOVA with Holm–Šídák multiple-comparisons testing; false discovery rate (FDR) correction (q = 0.05) was applied.

Snf1 is specifically and acutely required for MDC formation induced by mitochondrial biogenesis–activating conditions (related to Figure 3).

A. Quantification of MDC formation in PKA-pathway mutants following 2 h glucose restriction. B. Quantification of MDC formation in Snf1-associated mutants following 2 h glucose restriction. C. Immunoblot analysis of Snf1-AID-FLAG strains and controls following auxin treatment. Haploid parent strains, an untagged OsTIR1 control, and diploid Snf1-AID-FLAG strains were treated with DMSO or auxin for the indicated times. FLAG was detected by immunoblot; Ponceau staining serves as a loading control. D. Quantification of MDC formation in Snf1-AID strains following auxin-induced degradation and 2 h carbon-source switching to galactose. Cells were pretreated with DMSO or auxin for 30 min before the media shift. E. Quantification of MDC formation in Snf1-AID strains following auxin treatment and 2 h under trace-glucose conditions. Cells were pretreated with DMSO or auxin for 30 min before the media shift. F. Quantification of MDC formation in Snf1-AID strains following auxin treatment and 2 h exposure to 2-deoxy-D-glucose (2-DG). Cells were pretreated with DMSO or auxin for 30 min before 2-DG treatment. G. Quantification of MDC formation in Snf1-AID strains following auxin treatment and 2 h concanamycin A (ConcA) exposure. Cells were pretreated with DMSO or auxin for 30 min before ConcA treatment. H. Quantification of MDC formation following auxin pretreatment and glucose restriction. Control (Snf1-AID-FLAG + EV) and test (Snf1-AID-FLAG + His3-OsTIR1 + SCM4 OE) strains were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to 2 h glucose restriction. I. Quantification of MDC formation in EV control, SCM4 OE, snf1Δ, and snf1Δ + SCM4 OE strains following 2 h glucose restriction. Quantification and statistics. All MDC measurements were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

Analysis of Snf1-dependent pathways involved in MDC induction (related to Figure 4).

A. Quantification of MDC formation in mutants affecting Snf1-regulated and mitochondrial remodeling pathways following 2 h glucose restriction. B. Quantification of MDC formation following auxin pretreatment and glucose restriction. Control (Acc1-AID–FLAG) and test (Acc1-AID–FLAG + His3-OsTIR1) strains were grown overnight in YPAD, pretreated with DMSO or auxin for 30 min, and subjected to 2 h glucose restriction. C. Quantification of MDC formation in Acc1-AID strains following auxin treatment and 2 h rapamycin exposure. Cells were pretreated with DMSO or auxin for 30 min before rapamycin treatment. D. Quantification of MDC formation under combined treatments with DMSO, rapamycin (RAP), concanamycin A (ConcA), YPAD (control), or YPA (glucose restriction), with or without cerulenin pretreatment. Cells were pretreated with DMSO or cerulenin for 30 min followed by 2 h treatment. E. Immunoblot analysis of AID strains used in Figure 4F and G and panels B-C following auxin-induced degradation. FLAG was detected by immunoblot; Ponceau staining serves as a loading control. The asterisk indicates a nonspecific band. F. Quantification of MDC formation in CRISPR-generated Mig1 phospho-null mutants following 2 h glucose restriction. Haploid mutants were mated with mig1Δ strains prior to analysis. G. Immunoblot analysis of mitochondrial protein levels in wild-type cells following glucose restriction or NaCl stress in the presence of DMSO or thiolutin. Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. Quantification and statistics. All MDC measurements were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.

Mitochondrial protein expression and MDC regulation during distinct induction conditions (related to Figure 5).

A. Immunoblot analysis of mitochondrial protein levels in wild-type cells following 6 h treatment with rapamycin, ConcA, CHX, YPA, or S. Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. B. Immunoblot analysis of mitochondrial protein levels in Snf1-AID–FLAG strains following glucose restriction or rapamycin treatment with or without auxin-induced degradation. Om45 and Por1 are shown; Vph1 and Ponceau staining serve as loading controls. C. Quantification of MDC formation in Hap4 expression strains following rapamycin treatment. Cells were grown overnight in SD-URA with or without β-estradiol to induce sustained Hap4-FLAG expression and then treated with rapamycin for 2 h. Quantification and statistics. MDC measurements in (C) were performed with n = 3 independent experiments and 100 cells scored per replicate. Error bars represent mean ± SEM. Statistical significance was assessed using Welch’s two-tailed t-test.