Assays for studying normal versus suppressive ERAD-associated retrotranslocation pathways in yeast

STAR Protoc. 2021 Jul 7;2(3):100640. doi: 10.1016/j.xpro.2021.100640. eCollection 2021 Sep 17.

Abstract

In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1's role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).

Keywords: Cell Biology; Cell culture; Cell separation/fractionation; Cell-based Assays; Flow Cytometry/Mass Cytometry; Genetics; Model Organisms; Protein Biochemistry.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Endoplasmic Reticulum-Associated Degradation* / genetics
  • Endoplasmic Reticulum-Associated Degradation* / physiology
  • Gene Knockout Techniques / methods*
  • Membrane Proteins* / genetics
  • Membrane Proteins* / metabolism
  • Membrane Proteins* / physiology
  • Saccharomyces cerevisiae Proteins* / genetics
  • Saccharomyces cerevisiae Proteins* / metabolism
  • Saccharomyces cerevisiae Proteins* / physiology
  • Saccharomyces cerevisiae* / cytology
  • Saccharomyces cerevisiae* / genetics

Substances

  • Dfm1 protein, S cerevisiae
  • Membrane Proteins
  • Saccharomyces cerevisiae Proteins