A cycloheximide sensitivity factor from yeast required for N-acetylphenylalanylpuromycin formation

Area of Science:

• Molecular Biology
• Protein Synthesis

Background:

• Polyphenylalanine synthesis is a key process in protein translation.
• Cycloheximide is a known inhibitor of protein synthesis.
• The role of specific protein factors in modulating antibiotic sensitivity is an area of ongoing research.

Purpose of the Study:

• To isolate and characterize a protein factor from yeast that influences cycloheximide sensitivity.
• To elucidate the function of this factor in polyphenylalanine synthesis and related processes.

Main Methods:

• Isolation and partial purification of a protein factor (factor P) from yeast.
• Assay of polyphenylalanine synthesis in the presence and absence of factor P.
• Measurement of cycloheximide inhibition at various concentrations.
• Analysis of N-acetylphenylalanylpuromycin formation.
• Utilizing antibodies against elongation factor 2 (EF-2) to probe functional steps.

Main Results:

• Factor P significantly increased yeast polyphenylalanine synthesis sensitivity to cycloheximide (50% inhibition shifted from 10(-3) M to 10(-6) M).
• Factor P was essential for EF-2 dependent N-acetylphenylalanylpuromycin formation.
• The requirement for factor P in N-AcPhe-tRNA transfer to puromycin was demonstrated.
• Antibody studies indicated factor P acts after EF-2 translocation.

Conclusions:

• Factor P is a novel protein essential for conferring cycloheximide sensitivity to the yeast polyphenylalanine synthesis system.
• Factor P plays a critical role in the EF-2 dependent step of N-acetylphenylalanylpuromycin formation, subsequent to translocation.
• This finding provides new insights into the regulation of protein synthesis and antibiotic action in yeast.