The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Biology

Background:

• Protein degradation is crucial for cellular function.
• The 26 S proteasome, a large protein complex, degrades ubiquitinated proteins.
• This process requires ATP hydrolysis, mediated by six proteasomal ATPases.

Purpose of the Study:

• To investigate the functional mechanism of the six proteasomal ATPases.
• To determine if these ATPases operate independently or in a coordinated cyclic manner.
• To elucidate the relationship between ATP hydrolysis rate, substrate structure, and proteasome function.

Main Methods:

• Utilized yeast mutants with specific defects in ATP binding to proteasomal ATPase subunits (Rpt3, Rpt5, Rpt6).
• Measured basal and substrate-stimulated ATP hydrolysis rates.
• Quantified the degradation rates and energy expenditure for ubiquitinated dihydrofolate reductase (Ub5-DHFR) under varying conditions, including substrate folding and gate access.

Main Results:

• Single mutations in ATPase subunits significantly reduced basal ATP hydrolysis and abolished substrate-induced stimulation, indicating an ordered, cyclic function.
• Proteasome-mediated degradation of Ub5-DHFR showed a direct proportionality with ATP hydrolysis rate.
• Increased substrate (Ub5-DHFR) folding or reduced proteasome gate access increased degradation time and ATP consumption per molecule.

Conclusions:

• The six ATPase subunits of the 26 S proteasome function in an ordered, cyclic manner, with each subunit essential for substrate-induced activity.
• A fixed amount of ATP is consumed per ubiquitinated protein degraded.
• Polypeptide structure of the substrate influences the time and energy cost of proteasomal degradation, rather than the initial activation of the ATPase cycle.