Proteolytic activity of Yme1 is required for MDC formation.

(A) Widefield images of wild-type and yme1Δ cells expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (B) Quantification of (A) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (C) Widefield images of wild-type and yme1Δ cells endogenously tagged with Tom70-GFP and Tim50-mCherry expressing either EV or pRS413-Rpt3 (P215L) treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (D) Quantification of (C) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (E) Widefield images of wild-type and yme1Δ cells endogenously tagged with Tom70-GFP and Tim50-mCherry expressing either EV, pRS413-Yme1, or pRS413-Yme1 (E541Q) treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (F) Quantification of (E) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (G) Widefield images of yeast cells tagged with Tom70-GFP and Tim50-mCherry, constitutively overexpressing YME1 (YME1 OE), the protease-dead mutant, YME1(E541Q), or an empty vector (EV) control. White arrows denote MDCs. Scale bar = 2 µm. (H) Quantification of (G) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.

MDC Inducer Rapamycin triggers Yme1-dependent changes in the mitochondrial proteome.

(A) Clustered heatmap showing Z-scores of select mitochondrial proteins in wild-type and yme1Δ cells treated with DMSO or Rap for 2 h (n = 4), with corresponding mitochondrial subcategories indicated on the left. Proteins were categorized by GO Biological Processes (GO: BP) and manual annotation. Blue indicates upregulated proteins; red indicates downregulated proteins. (B) Volcano plot analysis of differentially expressed proteins in yme1Δ cells compared with wild-type cells following Rap treatment for 2 h (n = 4). Mitochondrial proteins enriched in yme1Δ cells are highlighted in blue and represent potential Yme1 substrates; red indicates downregulated proteins.

Yme1 regulation of Ups2 contributes to MDC formation.

(A) Immunoblot analysis of whole-cell lysates from yeast expressing endogenously tagged Ups1-HA, treated with DMSO or Rap for 2 h. Blots were probed with anti-HA; Pgk1 served as a loading control. Representative of n = 3 independent experiments. (B) Quantification of (A) wherein Ups1-HA levels were normalized to the corresponding Pgk1 signal and then normalized to EV cells treated with DMSO (n = 3). Statistical significance was determined by two-way ANOVA (*p ≤ 0.05, ***p ≤ 0.001). (C) Immunoblot analysis of whole-cell lysates from yeast expressing endogenously tagged Ups2-HA, treated with DMSO or Rap for 2 h. Blots were probed with anti-HA; Pgk1 served as a loading control. Representative of n = 3 independent experiments. (D) Quantification of (C) wherein Ups2-HA levels were normalized to the corresponding Pgk1 signal and then normalized to EV cells treated with DMSO (n = 3). Statistical significance was determined by two-way ANOVA (*p ≤ 0.05, **p ≤ 0.01). (E) Relative whole-cell PE levels in wild-type and yme1Δ cells treated with DMSO or Rap for 2 h. PE levels are normalized to total lipids. Error bars represent the mean ± SE of four biological replicates. Statistical significance was determined by two-way ANOVA with Holm-Šídák post hoc test comparing each condition to its corresponding DMSO control. (**p ≤ 0.01, ***p ≤ 0.001). (F) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (G) Quantification of (F) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.

MICOS complex contributes to Yme1-dependent regulation of MDC formation.

(A) Heatmap showing Z-scores of MICOS proteins in wild-type and yme1Δ cells treated with DMSO or Rap for 2 h (n = 4). Blue indicates upregulated proteins; red indicates downregulated proteins. (B) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (C) Quantification of (B) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (D) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry, grown in media containing low amino acids and treated with DMSO or ConcA for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (E) Quantification of (D) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (F) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (G) Quantification of (F) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.

Yme1-dependent MDC formation is gated by metabolic cues.

(A) Widefield images of yeast cells tagged with Tom70-GFP and Tim50-mCherry, constitutively overexpressing YME1 (YME1 OE), the protease-dead mutant, YME1(E541Q), or an empty vector (EV) control grown in minimal media that lacks amino acids. Scale bar = 2 µm. (B) Quantification of (A) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (C) Model of Yme1-mediated MDC biogenesis: Upon treatment with MDC stimulating stressors, Yme1 degrades Ups2, altering lipids, while also modulating the MICOS complex to possibly alter organizational or structural constraints that normally inhibit MDC formation. Panel C created with BioRender.com.

Secondary effects related to loss of Yme1 do not impact MDC formation (Related to Figure 1).

(A) Quantification of MDC formation in wild-type and yme1Δ cells treated with DMSO, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (B) Representative widefield images of distinct yeast mitochondrial morphologies. Scale bar = 2 µm. (C) Quantification of mitochondrial morphology in wild-type and yme1Δ cells expressing either EV or pRS413-Rpt3 (P215L). Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (D) Quantification of MDC formation in wild-type and yme1Δ cells expressing either EV or pRS413-Rpt3 (P215L) treated with DMSO, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (E) Quantification of mitochondrial morphology in wild-type and yme1Δ cells treated with DMSO or Rap for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (F) Widefield images of wild-type and rho0 cells expressing Tom70-GFP and Tim50-mCherry. White arrows denote MDCs. White arrows denote MDCs. Scale bar = 2 µm. (G) Quantification of (F) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (H) Quantification of MDC formation in wild-type and yme1Δ cells expressing either EV, pRS413-Yme1, or pRS413-Yme1 (E541Q) treated with DMSO, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.

Mitoproteomics data quality control (Related to Figure 2).

(A) Box plot showing log2-normalized protein intensities between wild-type and yme1Δ cells treated with DMSO or Rap for 2 h (n = 4). Comparable distributions across conditions confirm successful data normalization. (B) Principal component analysis of proteome data from wild-type and yme1Δ cells treated with DMSO or Rap for 2 h (n = 4).

MDC assay profiling of Yme1-regulated factors (Related to Figures 3 & 4).

(A) Quantification of MDC formation in wild-type and indicated mutants treated with DMSO, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (B) Quantification of MDC formation in wild-type and indicated mutants treated with DMSO, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (C) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (D) Quantification of (C) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (E) Quantification of MDC formation in wild-type and indicated mutants treated with DMSO, Rap, ConcA, or CHX for 2 h. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.

Role of the MICOS Complex in MDC formation: Effects of individual mutants and genetic interactions with Yme1, and Ups2 (Related to Figure 4).

(A) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry, grown in media containing low amino acids and treated with DMSO or ConcA for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (B) Quantification of (A) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate. (C) Tetrad dissection of mic60Δ/+ yme1Δ/+ diploid cells demonstrating synthetic lethality between MIC60 and YME1. (D) Widefield images of wild-type and indicated mutants expressing Tom70-GFP and Tim50-mCherry treated with DMSO or Rap for 2 h. White arrows denote MDCs. Scale bar = 2 µm. (E) Quantification of (D) showing the percentage of cells with MDCs. Error bars represent the mean ± SE of three replicates, n ≥ 100 cells per replicate.