Cell cycle-regulated proteolysis of mitotic target proteins

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Ubiquitin-dependent proteolysis regulates critical cell cycle transitions.
• The anaphase-promoting complex/cyclosome (APC/C) targets mitotic proteins for destruction.
• Understanding the regulation of protein degradation is key to cell cycle control.

Purpose of the Study:

• To investigate the regulated degradation of mitotic proteins (cyclins A and B, geminin H, Cut2p) across different cell cycle stages.
• To identify factors and motifs required for the destruction of these targets.
• To determine the conservation of these degradation mechanisms from yeast to humans.

Main Methods:

• Utilized cell-free extracts from mammalian somatic cells at various cell cycle stages (G0, G1, S, G2, M).
• Assessed the degradation of target proteins in vitro.
• Employed dominant-negative UbcH10 and Ubc3/Cdc34 in vitro and in vivo.

Main Results:

• All four investigated targets were degraded in G1 extracts but not in S phase extracts.
• Destruction requires an N-terminal destruction box motif and is dependent on UbcH10 activity.
• A PP2A-like phosphatase is necessary for G1 destruction, and a nuclear activity terminating destruction appears at S phase onset.
• Inhibition of UbcH10 or Ubc3/Cdc34 disrupted anaphase onset and chromosome congression, respectively.

Conclusions:

• Regulated ubiquitination and destruction of mitotic proteins are conserved from yeast to humans.
• The APC/C-UbcH10 machinery plays a vital role in controlling mitotic protein levels.
• Specific motifs and regulatory factors govern the timing and execution of protein degradation during the cell cycle.