Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways.

Area of Science:

• Protein quality control
• Molecular cell biology
• Ubiquitin-proteasome system

Background:

• Endoplasmic reticulum-associated degradation (ERAD) removes misfolded proteins from the ER.
• ERAD involves ubiquitylation by E3 ligases, retrotranslocation, and proteasomal degradation.
• Overexpression of ERAD factors is a common method to study ERAD.

Purpose of the Study:

• To investigate the impact of ERAD E3 ligase overexpression on substrate turnover in yeast.
• To identify limiting factors and off-pathway mechanisms in ERAD.

Main Methods:

• Analysis of ERAD E3 complex (Doa10, Hrd1, Asi1-3) overexpression in yeast.
• Assessment of substrate degradation and E2 enzyme (Ubc6) limitation.
• Utilizing dominant-negative ERAD mutants and examining Dfm1-deficient cells.

Main Results:

• Elevated Doa10 or Hrd1 RING activity inhibits specific Doa10 substrate degradation.
• Ubc6 (an ERAD E2 enzyme) becomes limiting upon ERAD E3 overexpression.
• Dominant-negative Doa10 mutants induce Ubc6 degradation independently of endogenous ERAD E3s and Dfm1.

Conclusions:

• Rogue RING activity at the ER membrane causes specific off-pathway defects in the Doa10 ERAD pathway.
• The study suggests an ERAD E3-independent retrotranslocation mechanism exists.