Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein.
Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD). To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide. As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system. Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes. Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle.
Lee
Robert J
RJ
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Liu
Chang-Wei
CW
Harty
Carol
C
McCracken
Ardythe A
AA
Latterich
Martin
M
Römisch
Karin
K
DeMartino
George N
GN
Thomas
Philip J
PJ
Brodsky
Jeffrey L
JL
D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004912:Erythrocytes / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex