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. 2021 Jul 20;10:e65484. doi: 10.7554/eLife.65484

Figure 1. Supersaturated nuclear-encoded mitochondrial proteins aggregate in the cytosol.

(A) SDS-PAGE analysis of aggregation assay fractions of WT yeast cells that overexpressed Atp2FLAG, Atp20FLAG, Cox8FLAG, pRip1FLAG, Qcr8FLAG, Cor1FLAG, Mas1FLAG, Cox12FLAG, and Qcr6FLAG for 3 hr when 2% galactose with 0.1% glucose was used as the carbon source. Pellet fractions after 4000 and 125,000× g centrifugation are indicated as P4k and P125k, respectively. The soluble fraction at 125,000× g is indicated as S125k. n = 3. (B) Ten-fold dilutions of WT cells that expressed metastable proteins or controls that were spotted on selective medium agar plates with galactose as the main carbon source at 28 and 37°C. (C) Total protein cell extract from WT yeast grown at 24°C and treated with 0, 5, 10, or 15 µM carbonyl cyanide m-chlorophenyl hydrazine (CCCP) for 30 min. (D) Quantification of pRip1, pSod2, and pMdh1 from (C). Quantified data are shown as mean ± SEM. n = 3. (E) SDS-PAGE analysis of aggregation assay fractions of yeast cells that were treated with 15 µM CCCP for 30 min, with 2% sucrose as the carbon source. (F) SDS-PAGE analysis of aggregation assay fractions of WT (pam16WT) and pam16-3 mutant yeast strains grown at 19°C and shifted to 37°C for 0, 3, or 5 hr, with 2% sucrose as the carbon source. In (A), (C), (E), and (F), the samples were separated by SDS-PAGE and identified by western blot with specific antisera. n = 3 for each experiment. *Nonspecific; p: presequence protein; i: intermediate protein; m: mature protein. *p<0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.

Figure 1—source data 1. Aggregation propensity characterization of mitochondrial proteins.
Protein sequences and information about mitochondrial targeting presequences were acquired from the Saccharomyces Genome Database and verified using Mitofates (Fukasawa et al., 2015) and MitoProt (Claros and Vincens, 1996) software. Protein solubility was analyzed using CamSol (Sormanni et al., 2015) software. Proteins and sequence residues with scores < –1 were poorly soluble and indicated as potential self-assembly hotspots (red). Scores > 1 characterize highly soluble proteins and sequence residues (blue). TMDs: transmembrane domains; IMS: intermembrane space; IM: inner membrane.
elife-65484-fig1-data1.docx (186.7KB, docx)

Figure 1.

Figure 1—figure supplement 1. Supersaturated nuclear-encoded mitochondrial protein aggregate in the cytosol and stimulate growth defects.

Figure 1—figure supplement 1.

(A) Schematic representation of the aggregation assay. (B, C) Metastable proteins aggregate in the cell, showing SDS-PAGE analysis of aggregation assay fractions of WT yeast cells that overexpressed Atp2FLAG and Atp20FLAG (B) and Cox8FLAG, Qcr8FLAG, pRip1FLAG, iRip1FLAG, mRip1FLAG, Cor1FLAG, Mas1FLAG, Cox12FLAG, and Qcr6FLAG (C) for 3 hr at 28°C, with 2% galactose with 0.1% glucose as the carbon source. (D) Metastable proteins exhibit a temperature-sensitive phenotype. Each protein-expressing strain was subjected to consecutive 10-fold dilutions and spotted on selective medium agar plates with glucose as the main carbon source at 28 and 37°C. (E) Cartoon representation of Rip1, Sod2, and Mdh1 mitochondrial proteins containing their presequences. (F) Total protein cell extract from WT (pam16WT) and pam16-3 mutant yeast strains that were grown at a permissive temperature of 19°C and shifted to a restrictive temperature of 37°C for 0, 1, 3, or 5 hr. In (B), (C), and (F), protein samples were separated by SDS-PAGE and identified by western blot with specific antisera. Each experiment was repeated three times. p: presequence protein; m: mature protein; i: intermediate; *: nonspecific; aa: amino acid.