To identify new components of the protein quality control and degradation pathway of the endoplasmic reticulum, we performed a growth-based genome wide screen of about 5000 viable deletion mutants of the yeast Saccharomyces cerevisiae. As substrates we used two misfolded ER membrane proteins: CTL* and Sec61-2L, chimeric derivatives of the classical ER degradation substrates CPY* and Sec61-2. Both substrates contain a cytosolic Leu2 protein fusion and stabilization of these substrates in ERAD deficient strains enables a restored growth of the transformed LEU2 deficient deletion mutants. We identified the strain deleted for the E4 ubiquitin ligase Hul5 among the mutant strains with a strong growth phenotype. Here we show that Hul5 is necessary for the degradation of two misfolded ER membrane substrates. While the degradation of their N-terminal parts is Hul5 independent, the breakdown of their C-terminal fragments requires the E4 activity of Hul5. In the absence of Hul5 a truncated form of CTL*myc remains to a large extent embedded in the ER membrane. Hul5 activity promotes the interaction of this truncated CTL*myc with the AAA-ATPase Cdc48, which is known to pull proteins out of the ER membrane. This study unravels the stepwise elimination of the ER membrane localized CTL*myc substrate. First, N-terminal, lumenal CPY* is transferred to the cytoplasm and degraded by the proteasome. Subsequently, the remaining C-terminal membrane anchored part requires Hul5 for its effective extraction out of the endoplasmic reticulum and proteasomal degradation.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|