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In vitro reconstitution of substrate dislocation from the endoplasmic reticulum using Perfringolysin O
Author(s) -
Sanyal Sumana
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.905.4
Subject(s) - endoplasmic reticulum , cytosol , endoplasmic reticulum associated protein degradation , cytoplasm , microbiology and biotechnology , in vitro , chemistry , biology , unfolded protein response , biophysics , biochemistry , enzyme
Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or “dislocated” into the cytosol for ER‐associated degradation (ERAD). To examine different roles of components in ERAD we have taken a biochemical approach to reconstitute the process of dislocation of misfolded substrates. Semi‐intact cells are prepared by treating with perfringolysin O (PFO), a pore forming toxin, for selective removal of the cytoplasm. We use a truncated version of ribophorin (RI 332 ) as model dislocation substrate. Cells expressing RI 332 are pulse‐labeled with 35 S; following treatment with PFO, cytosol is selectively removed from these cells, leaving the ER and other organelles intact. We find that RI 332 is quantitatively released from the ER and appears in the cytosolic fraction as the deglycosylated form due to activity of cytosolic PNGase F. Using this in vitro strategy we have been able to test various mutants of ER and cytosolic proteins to determine what affects dislocation of misfolded substrates. Yod1 is a deubiquitinating enzyme that has been shown to be involved in the dislocation of RI 332 . With a catalytically inactive mutant of Yod1, we find that the entire pool of RI 332 remains in the ER whereas with wild‐type Yod1, the deglycosylated version of RI 332 starts to appear in the cytosolic fraction in a time‐dependent manner.