Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.
We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA(Delta)), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER-Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA(Delta), although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Shuyu
S
Li
Robert A
RA
Spooner
Stuart C H
SC
Allen
Christopher P
CP
Guise
Graham
G
Ladds
Tina
T
Schnöder
Manfred J
MJ
Schmitt
J Michael
JM
Lord
Lynne M
LM
Roberts
D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D015723:Gene Library; D006056:Golgi Apparatus / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012276:Ricin / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D054875:Ubiquitination
D018832:Molecular Chaperones; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012276:Ricin; D046988:Proteasome Endopeptidase Complex; D008239:Lysine