Understanding the role of yeast Yme1 in mitochondrial finction using biochemical and proteomics analysis

ABSTRACT: Mitochondrial proteome comparison between S. cerevisiae BY4741 WT and Yme1 deletion mutant. Mitochondria were isolated by fractionation as described by Titorenko et al. (2009). Briefly, cells were grown in YPGal and harvested at later exponential phases of OD ~1.5. After pre- treatment with DTT buffer (100 mM Tris-H 2 SO 4 pH 9.4, 10 mM DTT) at 30°C for 20 minutes, the cell wall was digested with zymolyase 20T (Seikagaku Corporation, Japan) in zymolyase buffer (20mM potassium phosphate buffer pH 7.4, 1.2M sorbitol) for 1 hour at 30˚ with shaking at 70 rpm. The resulting spheroblasts were checked under a light microscope to confirm cell wall digestion. Then, the spheroblasts were lysed in homogenization buffer (20mM HEPES-KOH pH7.4, 0.6M sorbitol, 1mM phenylmethylsulfonyl fluoride (PMSF)) on- ice using a glass Teflon homogenizer. The resulting spheroblasts were subjected to two centrifugation steps at 1500 ×g to remove the cell debris and nuclei. Crude mitochondrial materials were isolated from the supernatant by centrifugation at 12,000 ×g at 4˚C for 10 minutes, then resuspended in SEM buffer (250mM sucrose, 0.6M Sorbitol, 20mM HEPES- KOH pH7.4). Protein concentration were checked by measuring absorbance at 280 nm, assuming an absorbance value of A280=0.21 would equate to 10 mg/ml of crude mitochondrial extract. After adjusting to final concentration of 10 mg/ml the crude mitochondria were then flash frozen and stored at −80°C.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Kwan Ting Kan  

LAB HEAD: Dr. Hui Lu

PROVIDER: PXD036367 | Pride | 2022-11-11

REPOSITORIES: pride