Abstract
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.
Highlights
Reticulum (ER)5 before they continue the journey to their final destination
To gain deeper insight into the components of ER protein quality control and degradation (ERQD) [39], a genome-wide screen was performed using the EUROSCARF yeast library consisting of about 5000 diploid S. cerevisiae strains with a leucine auxotrophy
Besides previously found ERQD components discovered in screens performed with CTL* [22, 27], we identified in addition the mutant strain deleted for the encoding sequence of Hul5
Summary
W303prc1-1 ⌬der3 ⌬pdr5 ⌬hul5 ⌬hul5⌬ubp6 ⌬ubc W303⌬prc1 ⌬prc1⌬der3 ⌬prc1⌬hul5 ⌬prc1⌬ubp6 ⌬prc1⌬hul5⌬ubp6 ⌬prc1⌬pdr5 ⌬prc1⌬pdr5⌬hul5 ⌬prc1⌬der3⌬hul UFD1 ufd. The screens are based on the stabilization of two misfolded ERAD membrane substrates, CTL* and Sec61-2L, both carrying a cytosolic 3-isopropylmalate dehydrogenase (Leu protein). Such stabilization can be observed by a restored growth of the transformed leucine auxotrophic strains of a yeast deletion library [22, 27, 39]. Hul is a HECT domain containing ubiquitin-protein ligase known to interact with the proteasome via its Rpn subunit and is involved in ubiquitin chain elongation (E4 activity) of proteasomal substrates [31]. The absence of Hul affects the interaction of Cdc with CTL*myc These results provide further information on the fine-tuning of the extraction and degradation process of misfolded ER membrane proteins
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