In eukaryotic cells, nearly one third of all newly synthesized proteins are translocated into the endoplasmic reticulum (ER). Inside the ER, proteins are post‐translationally modified and folded by molecular chaperones into their native states. Mutations, transcriptional/translational errors, aberrant post‐translational modifications, or misassembly can result in ER‐associated degradation (ERAD), a quality control pathway which recognizes and degrades misfolded proteins to prevent toxic gain‐of‐function phenotypes. Given the vast array of conformers that proteins adopt, it is not surprising that many diseases result from protein degradation by the ERAD pathway.The rate‐limiting step during ERAD is substrate retrotranslocation from the ER into the cytoplasm, which occurs prior to proteasome‐mediated degradation. Therefore, the retrotranslocation step can tune degradation efficiency and potentially be targeted to mitigate ERAD‐related diseases. Retrotranslocation is driven by the AAA‐ATPase Cdc48p in yeast (p97 in mammals), which extracts integral membrane proteins either directly from the ER membrane or through an as‐yet‐elusive retrotranslocation channel. Although integral membrane proteins have diverse membrane spanning domains and orientation, little is known about how the placement of a misfolded domain in the ER lumen versus the cytoplasm influences ERAD efficiency. We predict that extraction of a large ER lumenal domain will slow retrotranslocation kinetics.To test this hypothesis, a pair of model ERAD substrates were designed to contain the same truncated nucleotide‐binding domain from a well‐characterized ERAD substrate, Ste6p*, but on opposite sides of the ER membrane. We previously demonstrated that a soluble version of this domain, NBD2*, when expressed in the cytoplasm is degraded by the proteasome, and therefore can be used as a transposable degron (Guerriero et al., JBC, 2013). Our first model substrate, Chimera A*, is a dual pass membrane protein fused to NBD2* positioned in the cytoplasm. By contrast, Chimera N*, is a single pass membrane protein which deposits NBD2* in the ER lumen. Initial characterization confirms the proper topology of these substrates and proteasome‐dependent degradation. Moreover, the substrates rely on ER resident E3 ligases, Hrd1p and Doa10p, for efficient degradation. We are currently determining the relative dependence on Cdc48p for both degradation and retrotranslocation in order to better understand the energetic barriers to ERAD (Supported by NIH grants DK101584 to C.J.G. and GM075061 to J.L.B.).
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