Ubiquitin ligase Hul5 is required for fragment-specific substrate degradation in endoplasmic reticulum-associated degradation.
To identify new components of the protein quality control and degradation pathway of the endoplasmic reticulum (ER), 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 ER-associated degradation-deficient strains enables a restored growth of the transformed LEU2-deficient deletion mutants. We identified the strain deleted for the ubiquitin chain elongating 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. Although the degradation of their N-terminal parts is Hul5-independent, the breakdown of their C-terminal fragments requires the ubiquitin chain elongating ligase 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.
Kohlmann
Sonja
S
Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
Schäfer
Antje
A
Wolf
Dieter H
DH
D000251:Adenosine Triphosphatases / Q000737:chemistry; D018797:Cell Cycle Proteins / Q000737:chemistry; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000737:chemistry; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D003513:Cycloheximide; C468547:HUL5 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein