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In vitro analysis of ER-associated protein degradation

  • Author(s): Garza, Renee Marie
  • et al.
Abstract

Endoplasmic reticulum-associated degradation (ERAD) is part of the cellular protein quality control system. Substrates of protein quality control are usually ubiquitinated proteins that are degraded by the proteasome. Hrd1p, a principal ER ubiquitin ligase, mediates ERAD of proteins residing in the ER to those that are prevented from passing through the ER to final destinations in every other part of the cell. Protein quality control pathways not only degrade misfolded or mutant proteins. They are also involved in the regulated destruction of otherwise normal proteins. The ER-localized Hmg2p is an example of a natural substrate recognized and degraded by the protein quality control pathway ERAD and specifically by Hrd1p. HMG2 is one of two isozymes in yeast encoding the rate-limiting enzyme for the production of isoprenoids and sterols. At the intersection of sterol synthesis and ERAD, Hmg2p regulated degradation is unique for the study of two important pathways. We have developed an in vitro assay that allows testing both Hmg2p regulation-specific and ERAD hypotheses. In vitro Hmg2p ubiquitination was in agreement with established in vivo criteria. In vitro analysis was broadened to the study how Hmg2p, a multi-spanning transmembrane protein, is removed from the ER membrane (retrotranslocation). Studies revealed full-length Hmg2p is liberated into the cytosol after retrotranslocation and Rad23p and Dsk2p play an unexpected role in this step. Furthermore, reputed retrotranslocation channels were not found to have a role in Hmg2p retrotranslocation. In vivo examination of Hmg2p degradation uncovered geranylgeranyl pyrophosphate (GGPP) stimulates Hmg2p degradation. Future GGPP analysis includes its addition to in vitro assays described in this thesis

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