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Partial purification of cytosolic proteins which control yeast mitochondrial protein synthesis
Abstract:
Protein synthesis in isolated yeast mitochondria incubated in the presence of GTP is stimulated 2-fold by addition of dialyzed postpolysomal supernatant (S-150) at the start of the incubation. Incubation of the yeast S-150 with 5'-nucleotidase had no effect on the stimulatory activity suggesting that the increased protein synthesis does not result from guanine nucleotides. A partial purification of the protein factors which stimulate mitochondrial protein synthesis has been accomplished by chromatography on Sephacryl S-200. Stimulatory activity was eluted in two peaks, one in the 40,000 to 80,000 molecular weight range and a broad peak with a molecular weight of less than 10,000. Stimulation of mitochondrial protein synthesis by the low molecular weight activator fraction was proportional to the concentration of protein added and abolished by trypsin treatment suggesting that the low molecular weight activator is a protein(s). The rate of mitochondrial protein synthesis in the presence of activator, was linear for 40 min, while that in the presence of GTP was linear for only 20 min, suggesting that the activator and GTP stimulate protein synthesis by different mechanisms. Analysis of the products of the stimulated mitochondrial protein synthesis by gel electrophoresis revealed that the activator increased equally the labeling of all products. These results indicate that low molecular weight proteins present in the cytosol regulate mitochondrial protein synthesis.
Insights
Cytosolic proteins significantly enhance yeast mitochondrial protein synthesis, acting through a distinct mechanism compared to GTP. This finding reveals novel regulatory pathways for mitochondrial gene expression.
Area of Science:
- Mitochondrial Biology
- Molecular Genetics
- Biochemistry
Background:
- Mitochondrial protein synthesis is crucial for cellular respiration.
- Regulation of mitochondrial protein synthesis by cytosolic factors is not fully understood.
Purpose of the Study:
- To identify and characterize cytosolic factors that regulate protein synthesis in isolated yeast mitochondria.
- To elucidate the mechanism of action of these factors and compare it to GTP-stimulated synthesis.
Main Methods:
- Isolation of yeast mitochondria and incubation with GTP.
- Fractionation of postpolysomal supernatant (S-150) using Sephacryl S-200 chromatography.
- Enzymatic treatments (5'-nucleotidase, trypsin) to characterize the stimulatory factors.
- Analysis of protein synthesis products using gel electrophoresis.
Main Results:
- Dialyzed S-150 stimulated mitochondrial protein synthesis twofold.
- Stimulatory activity was found in two molecular weight fractions: 40-80 kDa and <10 kDa.
- The low molecular weight fraction, identified as protein(s), enhanced synthesis linearly for 40 min, unlike GTP's 20 min.
- The activator equally increased labeling of all mitochondrial protein products.
Conclusions:
- Low molecular weight cytosolic proteins act as potent stimulators of yeast mitochondrial protein synthesis.
- These proteins likely employ a different regulatory mechanism than GTP.
- This study uncovers a novel layer of cytosolic regulation over mitochondrial gene expression.